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Structure of the M2 muscarinic receptor-?-arrestin complex in a lipid nanodisc. Nature Staus, D. P., Hu, H., Robertson, M. J., Kleinhenz, A. L., Wingler, L. M., Capel, W. D., Latorraca, N. R., Lefkowitz, R. J., Skiniotis, G. 2020
Abstract

Following agonist activation, G-protein-coupled receptors (GPCRs) recruit ?-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis1. Additionally, ?-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins2. Here we present a cryo-electron microscopy (cryo-EM) structure of ?-arrestin 1 (?arr1) in complex with muscarinic acetylcholine-2-receptor (M2R) reconstituted in lipid nanodiscs. The M2R-?arr1 structure shows a multimodal network of flexible interactions, including binding of the ?arr1 N domain to phosphorylated receptor residues and ?arr1 finger loop insertion into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric Go protein structure3. Moreover, the cryoEM map reveals that the ?arr1 C-domain edge engages the lipid bilayer. Through atomistic simulations, biophysical, biochemical, and cellular assays, we show that the C-edge is critical for stable complex formation, ?arr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest the cooperative interactions of ?-arrestin with both the receptor and phospholipid bilayer contribute to its functional versatility.

View details for DOI 10.1038/s41586-020-1954-0

View details for PubMedID 31945772
Structure of the neurotensin receptor 1 in complex with ?-arrestin 1. Nature Huang, W., Masureel, M., Qianhui, Q., Janetzko, J., Inoue, A., Kato, H. E., Robertson, M. J., Nguyen, K. C., Glenn, J. S., Skiniotis, G., Kobilka, B. K. 2020
Abstract

Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization, and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information delineating the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-EM structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human ?-arrestin 1 (?arr1?CT). We found that phosphorylation of NTSR1 was critical for obtaining a stable complex with ?arr1?CT, and identified phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observed a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared to a structure of rhodopsin-arrestin-1, our structure displays an approximately 85� rotation of arrestin relative to the receptor. These findings highlight both conserved aspects but also the plasticity of arrestin-receptor interactions.

View details for DOI 10.1038/s41586-020-1953-1

View details for PubMedID 31945771
A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS. Nucleic acids research Yang, Y., Joshi, M., Takahashi, Y., Ning, Z., Qu, Q., Brunzelle, J. S., Skiniotis, G., Figeys, D., Shilatifard, A., Couture, J. 2019
Abstract

COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS's subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

View details for DOI 10.1093/nar/gkz1037

View details for PubMedID 31724694
Conformational transitions of a neurotensin receptor1-Gi1complex. Nature Kato, H. E., Zhang, Y., Hu, H., Suomivuori, C., Kadji, F. M., Aoki, J., Krishna Kumar, K., Fonseca, R., Hilger, D., Huang, W., Latorraca, N. R., Inoue, A., Dror, R. O., Kobilka, B. K., Skiniotis, G. 2019
Abstract

Neurotensin receptor1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3A. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts moreflexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

View details for DOI 10.1038/s41586-019-1337-6

View details for PubMedID 31243364
Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation. Molecular cell Gao, Y., Hu, H., Ramachandran, S., Erickson, J. W., Cerione, R. A., Skiniotis, G. 2019
Abstract

Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its alpha subunit (GalphaT). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GalphaT helical domain (alphaHD) contacting the Gbetagamma subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gbetagamma during receptor-catalyzed nucleotide exchange.

View details for DOI 10.1016/j.molcel.2019.06.007

View details for PubMedID 31300275
Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes SCIENCE Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G., Kobilka, B. K. 2019; 364 (6440): 552-+
View details for DOI 10.1126/science.aaw5188

View details for Web of Science ID 000467631800036
Structural insights into the activation of metabotropic glutamate receptors (vol 566, pg 79, 2019) NATURE Koehl, A., Hu, H., Feng, D., Sun, B., Zhang, Y., Robertson, M. J., Chu, M., Kobilka, T., Laeremans, T., Steyaert, J., Tarrasch, J., Dutta, S., Fonseca, R., Weis, W. I., Mathiesen, J. M., Skiniotis, G., Kobilka, B. K. 2019; 567 (7747): E10
View details for DOI 10.1038/s41586-019-0983-z

View details for Web of Science ID 000461126600009
Author Correction: Structural insights into the activation of metabotropic glutamate receptors. Nature Koehl, A., Hu, H., Feng, D., Sun, B., Zhang, Y., Robertson, M. J., Chu, M., Kobilka, T. S., Laeremans, T., Steyaert, J., Tarrasch, J., Dutta, S., Fonseca, R., Weis, W. I., Mathiesen, J. M., Skiniotis, G., Kobilka, B. K. 2019
Abstract

The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online.

View details for PubMedID 30814739
Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex CELL Kumar, K., Shalev-Benami, M., Robertson, M. J., Hu, H., Banister, S. D., Hollingsworth, S. A., Latorraca, N. R., Kato, H. E., Hilger, D., Maeda, S., Weis, W. I., Farrens, D. L., Dror, R. O., Malhotra, S. V., Kobilka, B. K., Skiniotis, G. 2019; 176 (3): 448-+
View details for DOI 10.1016/j.cell.2018.11.040

View details for Web of Science ID 000456526100007
Structural insights into the activation of metabotropic glutamate receptors. Nature Koehl, A., Hu, H., Feng, D., Sun, B., Zhang, Y., Robertson, M. J., Chu, M., Kobilka, T. S., Laermans, T., Steyaert, J., Tarrasch, J., Dutta, S., Fonseca, R., Weis, W. I., Mathiesen, J. M., Skiniotis, G., Kobilka, B. K. 2019
Abstract

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

View details for PubMedID 30675062
Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes. Science (New York, N.Y.) Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G., Kobilka, B. K. 2019; 364 (6440): 552?57
Abstract

Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/11 family, whereas M2R and M4R couple to the Gi/o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.

View details for PubMedID 31073061
Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex. Cell Krishna Kumar, K., Shalev-Benami, M., Robertson, M. J., Hu, H., Banister, S. D., Hollingsworth, S. A., Latorraca, N. R., Kato, H. E., Hilger, D., Maeda, S., Weis, W. I., Farrens, D. L., Dror, R. O., Malhotra, S. V., Kobilka, B. K., Skiniotis, G. 2018
Abstract

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), aG protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

View details for PubMedID 30639101
Structure and Conformational Dynamics of a COMPASS Histone H3K4 Methyltransferase Complex. Cell Qu, Q., Takahashi, Y., Yang, Y., Hu, H., Zhang, Y., Brunzelle, J. S., Couture, J., Shilatifard, A., Skiniotis, G. 2018
Abstract

The methylation of histone 3 lysine 4 (H3K4) is carried out by an evolutionarily conserved family of methyltransferases referred to as complex of proteins associated with Set1 (COMPASS). The activity of the catalytic SET domain (su(var)3-9, enhancer-of-zeste, and trithorax) is endowed through forming a complex with a set of core proteins that are widely shared from yeast to humans. We obtained cryo-electron microscopy (cryo-EM) maps of the yeast Set1/COMPASS core complex at overall 4.0- to 4.4-A resolution, providing insights into its structural organization and conformational dynamics. The Cps50 C-terminal tail weaves within the complex to provide a central scaffold for assembly. The SET domain, snugly positioned at the junction of the Y-shaped complex, is extensively contacted by Cps60 (Bre2), Cps50 (Swd1), and Cps30 (Swd3). The mobile SET-I motif of the SET domain is engaged by Cps30, explaining its key role in COMPASS catalytic activity toward higher H3K4 methylation states.

View details for PubMedID 30100186
Structure of the �-opioid receptor-Gi protein complex. Nature Koehl, A., Hu, H., Maeda, S., Zhang, Y., Qu, Q., Paggi, J. M., Latorraca, N. R., Hilger, D., Dawson, R., Matile, H., Schertler, G. F., Granier, S., Weis, W. I., Dror, R. O., Manglik, A., Skiniotis, G., Kobilka, B. K. 2018
Abstract

The mu-opioid receptor (muOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by muOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5A resolution cryo-electron microscopy structure of the muOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its Nterminus interacting with conserved receptor residues and its Cterminus engaging regions important for opioid-ligand selectivity. Comparison of the muOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein alpha-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the OR.

View details for PubMedID 29899455
Structural Basis for Teneurin Function in Circuit-Wiring: A Toxin Motif at the Synapse CELL Li, J., Shalev-Benami, M., Sando, R., Jiang, X., Kibrom, A., Wang, J., Leon, K., Katanski, C., Nazarko, O., Lu, Y. C., Sudhof, T. C., Skiniotis, G., Arac, D. 2018; 173 (3): 735-+
Abstract

Teneurins (TENs) are cell-surface adhesion proteins with critical roles in tissue development and axon guidance. Here, we report the 3.1-� cryoelectron microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc-toxins. The ECR includes a large ? barrel that partially encapsulates a C-terminal domain, which emerges to the solvent through an opening in the�mid-barrel region. An immunoglobulin (Ig)-like domain seals the bottom of the barrel while a ? propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the ? propeller acts as a switch to regulate trans-cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans-cellular signaling in a LPHN-dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.

View details for PubMedID 29677516
Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature Zhang, Y., Sun, B., Feng, D., Hu, H., Chu, M., Qu, Q., Tarrasch, J. T., Li, S., Sun Kobilka, T., Kobilka, B. K., Skiniotis, G. 2017; 546 (7657): 248-253
Abstract

Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein Gs. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1R-Gs complex at near atomic resolution. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the ?5-helix of the Ras-like domain of Gs. These results provide a structural framework for understanding class B GPCR activation through hormone binding.

View details for DOI 10.1038/nature22394

View details for PubMedID 28538729
Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature Liang, Y., Khoshouei, M., Radjainia, M., Zhang, Y., Glukhova, A., Tarrasch, J., Thal, D. M., Furness, S. G., Christopoulos, G., Coudrat, T., Danev, R., Baumeister, W., Miller, L. J., Christopoulos, A., Kobilka, B. K., Wootten, D., Skiniotis, G., Sexton, P. M. 2017; 546 (7656): 118-123
Abstract

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric G?s?? protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60� kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the ?5-helix of G?s. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the G? subunit. This structure provides a new framework for understanding G-protein-coupled receptor function.

View details for DOI 10.1038/nature22327

View details for PubMedID 28437792
Structural and Functional Analysis of a beta(2)-Adrenergic Receptor Complex with GRK5 Cell Komolov, K. E., Du, Y., Duc, N. M., Betz, R. M., Rodrigues, J. P., Leib, R. D., Patra, D., Skiniotis, G., Adams, C. M., Dror, R. O., Chung, K. Y., Kobilka, B. K., Benovic, J. L. 2017; 169 (3): 407-421 e16
Abstract

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK�complex remains poorly defined. We used a�comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ?2-adrenergic receptor (?2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in�the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ?2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism�by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

View details for DOI 10.1016/j.cell.2017.03.047

View details for PubMedID 28431242
Atomic resolution snapshot of Leishmania ribosome inhibition by the aminoglycoside paromomycin. Nature communications Shalev-Benami, M., Zhang, Y., Rozenberg, H., Nobe, Y., Taoka, M., Matzov, D., Zimmerman, E., Bashan, A., Isobe, T., Jaffe, C. L., Yonath, A., Skiniotis, G. 2017; 8 (1): 1589
Abstract

Leishmania is a single-celled eukaryotic parasite afflicting millions of humans worldwide, with current therapies limited to a poor selection of drugs that mostly target elements in the parasite's cell envelope. Here we determined the atomic resolution electron cryo-microscopy (cryo-EM) structure of the Leishmania ribosome in complex with paromomycin (PAR), a highly potent compound recently approved for treatment of the fatal visceral leishmaniasis (VL). The structure reveals the mechanism by which the drug induces its deleterious effects on the parasite. We further show that PAR interferes with several aspects of cytosolic translation, thus highlighting the cytosolic rather than the mitochondrial ribosome as the primary drug target. The results also highlight unique as well as conserved elements in the PAR-binding pocket that can serve as hotspots for the development of novel therapeutics.

View details for PubMedID 29150609

View details for PubMedCentralID PMC5693986
GPCR-G Protein-�-Arrestin Super-Complex Mediates Sustained G Protein Signaling. Cell Thomsen, A. R., Plouffe, B., Cahill, T. J., Shukla, A. K., Tarrasch, J. T., Dosey, A. M., Kahsai, A. W., Strachan, R. T., Pani, B., Mahoney, J. P., Huang, L., Breton, B., Heydenreich, F. M., Sunahara, R. K., Skiniotis, G., Bouvier, M., Lefkowitz, R. J. 2016; 166 (4): 907-919
Abstract

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ?-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some�GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ?-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ?-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ?-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

View details for DOI 10.1016/j.cell.2016.07.004

View details for PubMedID 27499021
Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies. Journal of the American Chemical Society Ehsan, M., Du, Y., Scull, N. J., Tikhonova, E., Tarrasch, J., Mortensen, J. S., Loland, C. J., Skiniotis, G., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2016; 138 (11): 3789?96
Abstract

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

View details for PubMedID 26966956
Visualization of arrestin recruitment by a G-protein-coupled receptor NATURE Shukla, A. K., Westfield, G. H., Xiao, K., Reis, R. I., Huang, L., Tripathi-Shukla, P., Qian, J., Li, S., Blanc, A., Oleskie, A. N., Dosey, A. M., Su, M., Liang, C., Gu, L., Shan, J., Chen, X., Hanna, R., Choi, M., Yao, X. J., Klink, B. U., Kahsai, A. W., Sidhu, S. S., Koide, S., Penczek, P. A., Kossiakoff, A. A., Woods, V. L., Kobilka, B. K., Skiniotis, G., Lefkowitz, R. J. 2014; 512 (7513): 218-?
Abstract

G-protein-coupled receptors (GPCRs) are critically regulated by ?-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ?2 adrenergic receptor (?2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ?-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-?-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ?2AR-?-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ?2AR and ?-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ?-arrestin 1 to the ?2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ?-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ?-arrestin 1 when coupled to the ?2AR. A molecular model of the ?2AR-?-arrestin signalling complex was made by docking activated ?-arrestin 1 and ?2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

View details for DOI 10.1038/nature13430

View details for Web of Science ID 000340200700037

View details for PubMedID 25043026

View details for PubMedCentralID PMC4134437
Structure of a modular polyketide synthase NATURE Dutta, S., Whicher, J. R., Hansen, D. A., Hale, W. A., Chemler, J. A., Congdon, G. R., Narayan, A. R., Hakansson, K., Sherman, D. H., Smith, J. L., Skiniotis, G. 2014; 510 (7506): 512-?
Abstract

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

View details for DOI 10.1038/nature13423

View details for Web of Science ID 000337806300041

View details for PubMedID 24965652

View details for PubMedCentralID PMC4278352
Structural rearrangements of a polyketide synthase module during its catalytic cycle NATURE Whicher, J. R., Dutta, S., Hansen, D. A., Hale, W. A., Chemler, J. A., Dosey, A. M., Narayan, A. R., Hakansson, K., Sherman, D. H., Smith, J. L., Skiniotis, G. 2014; 510 (7506): 560-?
Abstract

The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the ?-keto intermediate, and after ?-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.

View details for DOI 10.1038/nature13409

View details for Web of Science ID 000337806300052

View details for PubMedID 24965656

View details for PubMedCentralID PMC4074775
Ligand-Induced Architecture of the Leptin Receptor Signaling Complex MOLECULAR CELL Mancour, L. V., Daghestani, H. N., Dutta, S., Westfield, G. H., Schilling, J., Oleskie, A. N., Herbstman, J. F., Chou, S. Z., Skiniotis, G. 2012; 48 (4): 655-661
Abstract

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.

View details for DOI 10.1016/j.molcel.2012.09.003

View details for Web of Science ID 000311919500017

View details for PubMedID 23063524

View details for PubMedCentralID PMC3513567
Crystal structure of the beta(2) adrenergic receptor-Gs protein complex NATURE Rasmussen, S. G., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., Mathiesen, J. M., Shah, S. T., Lyons, J. A., Caffrey, M., Gellman, S. H., Steyaert, J., Skiniotis, G., Weis, W. I., Sunahara, R. K., Kobilka, B. K. 2011; 477 (7366): 549-U311
Abstract

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ?(2) adrenergic receptor (?(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ?(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ?(2)AR and Gs involve the amino- and carboxy-terminal ?-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ?(2)AR include a 14 � outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an ?-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the ?-helical domain of G?s relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

View details for DOI 10.1038/nature10361

View details for Web of Science ID 000295320900031

View details for PubMedID 21772288

View details for PubMedCentralID PMC3184188
Structural flexibility of the Gas alpha-helical domain in the beta(2)-adrenoceptor Gs complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Westfield, G. H., Rasmussen, S. G., Su, M., Dutta, S., DeVree, B. T., Chung, K. Y., Calinski, D., Velez-Ruiz, G., Oleskie, A. N., Pardon, E., Chae, P. S., Liu, T., Li, S., Woods, V. L., Steyaert, J., Kobilka, B. K., Sunahara, R. K., Skiniotis, G. 2011; 108 (38): 16086-16091
Abstract

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied ?(2)-adrenoceptor (?(2)AR) in complex with the heterotrimeric G protein Gs (G?s??). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the ?-helical domain of G?s appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the ?-helical domain on the Ras-like domain of G?s. Molecular modeling of the ?-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of G?s-GTP?S. These data argue that the ?-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

View details for DOI 10.1073/pnas.1113645108

View details for Web of Science ID 000295030000081

View details for PubMedID 21914848

View details for PubMedCentralID PMC3179071
Ribosome Assembly Factors Prevent Premature Translation Initiation by 40S Assembly Intermediates SCIENCE Strunk, B. S., Loucks, C. R., Su, M., Vashisth, H., Cheng, S., Schilling, J., Brooks, C. L., Karbstein, K., Skiniotis, G. 2011; 333 (6048): 1449-1453
Abstract

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we present the electron cryo-microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae at 18 angstrom resolution. We obtained cryo-EM reconstructions of preribosomal complexes lacking individual components to define the positions of all seven AFs bound to this intermediate. These late-binding AFs are positioned to prevent each step in the translation initiation pathway. Together, they obstruct the binding sites for initiation factors, prevent the opening of the messenger RNA channel, block 60S subunit joining, and disrupt the decoding site. These redundant mechanisms probably ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation.

View details for DOI 10.1126/science.1208245

View details for Web of Science ID 000294672200043

View details for PubMedID 21835981

View details for PubMedCentralID PMC3402165
Structural organization of a full-length gp130/LIF-R cytokine receptor transmembrane complex MOLECULAR CELL Skiniotis, G., Lupardus, P. J., Martick, M., Walz, T., Garcia, K. C. 2008; 31 (5): 737-748
Abstract

gp130 is a shared receptor for at least nine cytokines and can signal either as a homodimer or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here, we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-Ralpha). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6Ralpha hexameric complex, CNTF/CNTF-Ralpha heterodimerizes gp130 and LIF-R via noncooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic analysis of the full-length gp130/LIF-R/CNTF-Ralpha/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the "tall" class of gp130 family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

View details for DOI 10.1016/j.molcel.2008.08.011

View details for Web of Science ID 000259113800014

View details for PubMedID 18775332

View details for PubMedCentralID PMC2607196
Structure of a VEGF-VEGF receptor complex determined by electron microscopy NATURE STRUCTURAL & MOLECULAR BIOLOGY Ruch, C., Skiniotis, G., Steinmetz, M. O., Walz, T., Ballmer-Hofer, K. 2007; 14 (3): 249-250
Abstract

Receptor tyrosine kinases are activated upon ligand-induced dimerization. Here we show that the monomeric extracellular domain of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) has a flexible structure. Binding of VEGF to membrane-distal immunoglobulin-like domains causes receptor dimerization and promotes further interaction between receptor monomers through the membrane-proximal immunoglobulin-like domain 7. By this mechanism, ligand-induced dimerization of VEGFR-2 can be communicated across the membrane, activating the intracellular tyrosine kinase domains.

View details for DOI 10.1038/nsmb1202

View details for Web of Science ID 000244715200017

View details for PubMedID 17293873
Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor NATURE STRUCTURAL & MOLECULAR BIOLOGY Skiniotis, G., Boulanger, M. J., Garcia, K. C., Walz, T. 2005; 12 (6): 545-551
Abstract

gp130 is a shared cytokine signaling receptor and the founding member of the 'tall' class of cytokine receptors. A crystal structure of the ligand-binding domains of gp130 in complex with human interleukin-6 (IL-6) and its a-receptor (IL-6Ralpha) revealed a hexameric architecture in which the gp130 membrane-distal regions were approximately 100 A apart, in contrast to the close apposition seen between short cytokine receptor complexes. Here we used single-particle EM to visualize the entire extracellular hexameric IL-6-IL-6Ralpha-gp130 complex, containing all six gp130 domains. The structure reveals that gp130 is bent such that the membrane-proximal domains of gp130 are close together at the cell surface, enabling activation of intracellular signaling. Variation in the receptor bend angles suggests a possible conformational transition from open to closed states upon ligand binding; this transition is probably representative of the other tall cytokine receptors.

View details for DOI 10.1038/nsmb941

View details for Web of Science ID 000229533800016

View details for PubMedID 15895091
CRYO-EM Structures of the GIRK2 Channel Reveal Mechanisms for Lipid Modulation Glaaser, I. W., Mathiharan, Y. K., Zhao, Y., Skiniotis, G., Slesinger, P. A. CELL PRESS. 2020: 497A?498A
View details for Web of Science ID 000513023203234
Structures of G? Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms. Cell reports Seven, A. B., Hilger, D., Papasergi-Scott, M. M., Zhang, L., Qu, Q., Kobilka, B. K., Tall, G. G., Skiniotis, G. 2020
Abstract

Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of G? subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-G?i and Ric-8A-G?q complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of G?, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal ?5 helix of G? is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the G? C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal ?5 helix before allowing GTP-gated release of G? from Ric-8A.

View details for DOI 10.1016/j.celrep.2020.02.086

View details for PubMedID 32126208
Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability. Chemistry (Weinheim an der Bergstrasse, Germany) Ehsan, M., Du, Y., Mortensen, J. S., Hariharan, P., Qu, Q., Ghani, L., Das, M., Grethen, A., Byrne, B., Skiniotis, G., Keller, S., Loland, C. J., Guan, L., Kobilka, B. K., Chae, P. S. 2019
Abstract

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-beta-d-maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human beta2 adrenergic receptor (beta2 AR), a G-protein coupled receptor, and its complex with Gs protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.

View details for DOI 10.1002/chem.201902468

View details for PubMedID 31243822
De novo computational RNA modeling into cryo-EM maps of large ribonucleoprotein complexes. Nature methods Kappel, K., Liu, S., Larsen, K. P., Skiniotis, G., Puglisi, E. V., Puglisi, J. D., Zhou, Z. H., Zhao, R., Das, R. 2018
Abstract

Increasingly, cryo-electron microscopy (cryo-EM) is used to determine the structures of RNA-protein assemblies, but nearly all maps determined with this method have biologically important regions where the local resolution does not permit RNA coordinate tracing. To address these omissions, we present de novo ribonucleoprotein modeling in real space through assembly of fragments together with experimental density in Rosetta (DRRAFTER). We show that DRRAFTER recovers near-native models for a diverse benchmark set of RNA-protein complexes including the spliceosome, mitochondrial ribosome, and CRISPR-Cas9-sgRNA complexes; rigorous blind tests include yeast U1 snRNP and spliceosomal P complex maps. Additionally, to aid in model interpretation, we present a method for reliable in situ estimation of DRRAFTER model accuracy. Finally, we apply DRRAFTER to recently determined maps of telomerase, the HIV-1 reverse transcriptase initiation complex, and the packaged MS2 genome, demonstrating the acceleration of accurate model building in challenging cases.

View details for PubMedID 30377372
Structural Analysis of the Ash2L/Dpy-30 Complex Reveals a Heterogeneity in H3K4 Methylation. Structure (London, England : 1993) Haddad, J. F., Yang, Y., Takahashi, Y., Joshi, M., Chaudhary, N., Woodfin, A. R., Benyoucef, A., Yeung, S., Brunzelle, J. S., Skiniotis, G., Brand, M., Shilatifard, A., Couture, J. 2018
Abstract

Dpy-30 is a regulatory subunit controlling the histone methyltransferase activity of the KMT2 enzymes invivo. Paradoxically, invitro methyltransferase assays revealed that Dpy-30 only modestly participates in the positive heterotypic allosteric regulation of these methyltransferases. Detailed genome-wide, molecular and structural studies reveal that an extensive network of interactions taking place at the interface between Dpy-30 and Ash2L are critical for the correct placement, genome-wide, of H3K4me2 and H3K4me3 but marginally contribute to the methyltransferase activity of KMT2 enzymes invitro. Moreover, we show that H3K4me2 peaks persisting following the loss of Dpy-30 are found in regions of highly transcribed genes, highlighting an interplay between Complex of Proteins Associated with SET1 (COMPASS) kinetics and the cycling of RNA polymerase to control H3K4 methylation. Overall, our data suggest that Dpy-30 couples its modest positive heterotypic allosteric regulation of KMT2 methyltransferase activity with its ability to help the positioning of SET1/COMPASS to control epigenetic signaling.

View details for PubMedID 30270175
Development of an antibody fragment that stabilizes GPCR/G-protein complexes. Nature communications Maeda, S., Koehl, A., Matile, H., Hu, H., Hilger, D., Schertler, G. F., Manglik, A., Skiniotis, G., Dawson, R. J., Kobilka, B. K. 2018; 9 (1): 3712
Abstract

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Galpha and Gbetagamma subunits in the heterotrimer, and confers resistance to GTPgammaS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.

View details for PubMedID 30213947
Vitamin E-based glycoside amphiphiles for membrane protein structural studies. Organic & biomolecular chemistry Ehsan, M., Du, Y., Molist, I., Seven, A. B., Hariharan, P., Mortensen, J. S., Ghani, L., Loland, C. J., Skiniotis, G., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2018; 16 (14): 2489?98
Abstract

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., ?-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile-lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.

View details for PubMedID 29564464
Architecture of an HIV-1 reverse transcriptase initiation complex. Nature Larsen, K. P., Mathiharan, Y. K., Kappel, K., Coey, A. T., Chen, D. H., Barrero, D., Madigan, L., Puglisi, J. D., Skiniotis, G., Puglisi, E. V. 2018
Abstract

Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection 1 and a common target of antiretroviral drugs 2 . The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA 5 each bound�to host lysine 3 transfer RNA (tRNALys3), which acts as a primer for initiation of reverse transcription6,7. Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

View details for PubMedID 29695867
Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel. Structure (London, England : 1993) Zhang, Y., Hong, S., Ruangprasert, A., Skiniotis, G., Dunham, C. M. 2018; 26 (3): 437?45.e3
Abstract

Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site are�observed. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ?54�� away from the 30S E site to interact with the 30S head domain and 50S uL5. This position of the E-site tRNA causes the uL1 stalk to adopt a more open conformation that likely represents an intermediate state during E-site tRNA dissociation. These results suggest that structured mRNAs at the entrance channel restrict 30S subunit movement required during translation to slow E-site tRNA dissociation.

View details for DOI 10.1016/j.str.2018.01.013

View details for PubMedID 29456023

View details for PubMedCentralID PMC5842130
Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study CHEMICAL SCIENCE Sadaf, A., Du, Y., Santillan, C., Mortensen, J. S., Molist, I., Seven, A. B., Hariharan, P., Skiniotis, G., Loland, C. J., Kobilka, B. K., Guan, L., Byrne, B., Chae, P. 2017; 8 (12): 8315?24
Abstract

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human ?2 adrenergic receptor (?2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of ?2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

View details for PubMedID 29619178

View details for PubMedCentralID PMC5858085
Mechanism of Vps4 hexamer function revealed by cryo-EM. Science advances Su, M., Guo, E. Z., Ding, X., Li, Y., Tarrasch, J. T., Brooks, C. L., Xu, Z., Skiniotis, G. 2017; 3 (4)
Abstract

Vps4 is a member of AAA+ ATPase (adenosine triphosphatase associated with diverse cellular activities) that operates as an oligomer to disassemble ESCRT-III (endosomal sorting complex required for transport III) filaments, thereby catalyzing the final step in multiple ESCRT-dependent membrane remodeling events. We used electron cryo-microscopy to visualize oligomers of a hydrolysis-deficient Vps4 (vacuolar protein sorting-associated protein 4) mutant in the presence of adenosine 5'-triphosphate (ATP). We show that Vps4 subunits assemble into an asymmetric hexameric ring following an approximate helical path that sequentially stacks substrate-binding loops along the central pore. The hexamer is observed to adopt an open or closed ring configuration facilitated by major conformational changes in a single subunit. The structural transition of the mobile Vps4 subunit results in the repositioning of its substrate-binding loop from the top to the bottom of the central pore, with an associated translation of 33 �. These structures, along with mutant-doping experiments and functional assays, provide evidence for a sequential and processive ATP hydrolysis mechanism by which Vps4 hexamers disassemble ESCRT-III filaments.

View details for DOI 10.1126/sciadv.1700325

View details for PubMedID 28439563

View details for PubMedCentralID PMC5392032
Distinct conformations of GPCR-beta-arrestin complexes mediate desensitization, signaling, and endocytosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cahill, T. J., Thomson, A. R., Tarrasch, J. T., Plouffe, B., Nguyen, A. H., Yang, F., Huang, L., Kahsai, A. W., Bassoni, D. L., Gavino, B. J., Lamerdin, J. E., Triest, S., Shukla, A. K., Berger, B., Little, J., Antar, A., Blanc, A., Qu, C., Chen, X., Kawakami, K., Inoue, A., Aoki, J., Steyaert, J., Sun, J., Bouvier, M., Skiniotis, G., Lefkowitz, R. J. 2017; 114 (10): 2562-2567
Abstract

?-Arrestins (?arrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-?arr complexes: the "tail" conformation, with ?arr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, ?arr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of ?arrs is unknown. Here, we created a mutant form of ?arr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and ?arr signaling but not desensitization of G protein signaling. Thus, the two GPCR-?arr conformations can carry out distinct functions.

View details for DOI 10.1073/pnas.1701529114

View details for Web of Science ID 000395511400062

View details for PubMedID 28223524

View details for PubMedCentralID PMC5347553
Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study: Implications of Detergent Kink for Micellar Properties. Journal of the American Chemical Society Das, M., Du, Y., Ribeiro, O., Hariharan, P., Mortensen, J. S., Patra, D., Skiniotis, G., Loland, C. J., Guan, L., Kobilka, B. K., Byrne, B., Chae, P. S. 2017; 139 (8): 3072-3081
Abstract

Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties. Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n-dodecyl-?-d-maltoside (DDM) for all membrane proteins tested. Efficacy of the individual NBMs varied depending on the overall detergent shape and alkyl chain length. Specifically, NBMs with no kink in the lipophilic region conferred greater stability to the proteins than NBMs with a kink. In addition, long alkyl chain NBMs were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes novel maltoside detergents with enhanced protein-stabilizing properties but also suggests that overall detergent geometry has an important role in determining membrane protein stability. Notably, this is the first systematic study on the effect of detergent kinking on micellar properties and associated membrane protein stability.

View details for DOI 10.1021/jacs.6b11997

View details for PubMedID 28218862
The Vps13p-Cdc31p complex is directly required for TGN late endosome transport and TGN homotypic fusion JOURNAL OF CELL BIOLOGY De, M., Oleskie, A. N., Ayyash, M., Dutta, S., Mancour, L., Abazeed, M. E., Brace, E. J., Skiniotis, G., Fuller, R. S. 2017; 216 (2): 425-439
Abstract

Yeast VPS13 is the founding member of a eukaryotic gene family of growing interest in cell biology and medicine. Mutations in three of four human VPS13 genes cause autosomal recessive neurodegenerative or neurodevelopmental disease, making yeast Vps13p an important structural and functional model. Using cell-free reconstitution with purified Vps13p, we show that Vps13p is directly required both for transport from the trans-Golgi network (TGN) to the late endosome/prevacuolar compartment (PVC) and for TGN homotypic fusion. Vps13p must be in complex with the small calcium-binding protein Cdc31p to be active. Single-particle electron microscopic analysis of negatively stained Vps13p indicates that this 358-kD protein is folded into a compact rod-shaped density (20 � 4 nm) with a loop structure at one end with a circular opening ?6 nm in diameter. Vps13p exhibits ATP-stimulated binding to yeast membranes and specific interactions with phosphatidic acid and phosphorylated forms of phosphatidyl inositol at least in part through the binding affinities of conserved N- and C-terminal domains.

View details for DOI 10.1083/jcb.201606078

View details for Web of Science ID 000394244800017

View details for PubMedID 28122955

View details for PubMedCentralID PMC5294781
Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex. The Journal of biological chemistry Gao, Y., Westfield, G., Erickson, J. W., Cerione, R. A., Skiniotis, G., Ramachandran, S. 2017; 292 (34): 14280?89
Abstract

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component ?T-GTP and ?1?1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a G?T/G?i1 chimera (?T*) and ?1?1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to G?T* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ?2-adrenergic receptor-GS complex, including a flexible ?T* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.

View details for DOI 10.1074/jbc.M117.797100

View details for PubMedID 28655769

View details for PubMedCentralID PMC5572916
Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on V?9V?2 T cell activation. Proceedings of the National Academy of Sciences of the United States of America Gu, S., Sachleben, J. R., Boughter, C. T., Nawrocka, W. I., Borowska, M. T., Tarrasch, J. T., Skiniotis, G., Roux, B., Adams, E. J. 2017; 114 (35): E7311?E7320
Abstract

Human V?9V?2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of V?9V?2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the V?9V?2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.

View details for DOI 10.1073/pnas.1707547114

View details for PubMedID 28807997

View details for PubMedCentralID PMC5584448
A snapshot of cryo-EM PROTEIN SCIENCE Skiniotis, G. 2017; 26 (1): 5-7
View details for DOI 10.1002/pro.3088

View details for Web of Science ID 000393961700001

View details for PubMedID 28024108

View details for PubMedCentralID PMC5192971
Structural Basis for Regulated Proteolysis by the ?-Secretase ADAM10. Cell Seegar, T. C., Killingsworth, L. B., Saha, N., Meyer, P. A., Patra, D., Zimmerman, B., Janes, P. W., Rubinstein, E., Nikolov, D. B., Skiniotis, G., Kruse, A. C., Blacklow, S. C. 2017; 171 (7): 1638?48.e7
Abstract

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic ?-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to�the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

View details for PubMedID 29224781

View details for PubMedCentralID PMC5773094
New penta-saccharide-bearing tripod amphiphiles for membrane protein structure studies. The Analyst Ehsan, M., Ghani, L., Du, Y., Hariharan, P., Mortensen, J. S., Ribeiro, O., Hu, H., Skiniotis, G., Loland, C. J., Guan, L., Kobilka, B. K., Byrne, B., Chae, P. S. 2017; 142 (20): 3889?98
Abstract

Integral membrane proteins either alone or as complexes carry out a range of key cellular functions. Detergents are indispensable tools in the isolation of membrane proteins from biological membranes for downstream studies. Although a large number of techniques and tools, including a wide variety of detergents, are available, purification and structural characterization of many membrane proteins remain challenging. In the current study, a new class of tripod amphiphiles bearing two different penta-saccharide head groups, designated TPSs, were developed and evaluated for their ability to extract and stabilize a range of diverse membrane proteins. Variations in the structures of the detergent head and tail groups allowed us to prepare three sets of the novel agents with distinctive structures. Some TPSs (TPS-A8 and TPS-E7) were efficient at extracting two proteins in a functional state while others (TPS-E8 and TPS-E10L) conferred marked stability to all membrane proteins (and membrane protein complexes) tested here compared to a conventional detergent. Use of TPS-E10L led to clear visualization of a receptor-Gs complex using electron microscopy, indicating profound potential in membrane protein research.

View details for DOI 10.1039/c7an01168g

View details for PubMedID 28913526

View details for PubMedCentralID PMC5818263
Extended surface for membrane association in Zika virus NS1 structure NATURE STRUCTURAL & MOLECULAR BIOLOGY Brown, W. C., Akey, D. L., Konwerski, J. R., Tarrasch, J. T., Skiniotis, G., Kuhn, R. J., Smith, J. L. 2016; 23 (9): 865-867
Abstract

The Zika virus, which has been implicated in an increase in neonatal microcephaly and Guillain-Barr� syndrome, has spread rapidly through tropical regions of the world. The virulence protein NS1 functions in genome replication and host immune-system modulation. Here, we report the crystal structure of full-length Zika virus NS1, revealing an elongated hydrophobic surface for membrane association and a polar surface that varies substantially among flaviviruses.

View details for DOI 10.1038/nsmb.3268

View details for Web of Science ID 000382712700016

View details for PubMedID 27455458
Flexible, symmetry-directed approach to assembling protein cages PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sciore, A., Su, M., Koldewey, P., Eschweiler, J. D., Diffley, K. A., Linhares, B. M., Ruotolo, B. T., Bardwell, J. C., Skiniotis, G., Marsh, E. N. 2016; 113 (31): 8681-8686
Abstract

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.

View details for DOI 10.1073/pnas.1606013113

View details for Web of Science ID 000380586600048

View details for PubMedID 27432965

View details for PubMedCentralID PMC4978231
2.8-angstrom Cryo-EM Structure of the Large Ribosomal Subunit from the Eukaryotic Parasite Leishmania CELL REPORTS Shalev-Benami, M., Zhang, Y., Matzov, D., Halfon, Y., Zackay, A., Rozenberg, H., Zimmerman, E., Bashan, A., Jaffe, C. L., Yonath, A., Skiniotis, G. 2016; 16 (2): 288-294
Abstract

Leishmania is a single-cell eukaryotic parasite of the Trypanosomatidae family, whose members cause an array of tropical diseases. The often fatal outcome of infections, lack of effective vaccines, limited selection of therapeutic drugs, and emerging resistant strains, underline the need to develop strategies to combat these pathogens. The Trypanosomatid ribosome has recently been highlighted as a promising therapeutic target due to structural features that are distinct from other eukaryotes. Here, we present the 2.8-� resolution structure of the Leishmania donovani large ribosomal subunit (LSU) derived from a cryo-EM map, further enabling the structural observation of eukaryotic rRNA modifications that play a significant role in ribosome assembly and function. The structure illustrates the unique fragmented nature of leishmanial LSU rRNA and highlights the irregular distribution of rRNA modifications in Leishmania, a characteristic with implications for anti-parasitic drug development.

View details for DOI 10.1016/j.celrep.2016.06.014

View details for Web of Science ID 000380262300002

View details for PubMedID 27373148

View details for PubMedCentralID PMC5835689
Highly Branched Pentasaccharide-Bearing Amphiphiles for Membrane Protein Studies JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Ehsan, M., Du, Y., Scull, N. J., Tikhonova, E., Tarrasch, J., Mortensen, J. S., Loland, C. J., Skiniotis, G., Guan, L., Byrne, B., Kobilka, B. K., Chae, P. S. 2016; 138 (11): 3789-3796
Abstract

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

View details for DOI 10.1021/jacs.5b13233

View details for Web of Science ID 000372854200027
Single-particle cryo-electron microscopy of macromolecular complexes MICROSCOPY Skiniotis, G., Southworth, D. R. 2016; 65 (1): 9-22
Abstract

Recent technological breakthroughs in image acquisition have enabled single-particle cryo-electron microscopy (cryo-EM) to achieve near-atomic resolution structural information for biological complexes. The improvements in image quality coupled with powerful computational methods for sorting distinct particle populations now also allow the determination of compositional and conformational ensembles, thereby providing key insights into macromolecular function. However, the inherent instability and dynamic nature of biological assemblies remain a tremendous challenge that often requires tailored approaches for successful implementation of the methodology. Here, we briefly describe the fundamentals of single-particle cryo-EM with an emphasis on covering the breadth of techniques and approaches, including low- and high-resolution methods, aiming to illustrate specific steps that are crucial for obtaining structural information by this method.

View details for DOI 10.1093/jmicro/dfv366

View details for Web of Science ID 000370062800003

View details for PubMedID 26611544

View details for PubMedCentralID PMC5895108
Structures of two distinct conformations of holo-non-ribosomal peptide synthetases NATURE Drake, E. J., Miller, B. R., Shi, C., Tarrasch, J. T., Sundlov, J. A., Allen, C. L., Skiniotis, G., Aldrich, C. C., Gulick, A. M. 2016; 529 (7585): 235-U289
Abstract

Many important natural products are produced by multidomain non-ribosomal peptide synthetases (NRPSs). During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighbouring catalytic domains in an assembly line fashion. Understanding the structural basis for catalysis with non-ribosomal peptide synthetases will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-non-ribosomal peptide synthetase modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and the single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering of novel non-ribosomal peptide synthetases.

View details for DOI 10.1038/nature16163

View details for Web of Science ID 000368015700042

View details for PubMedID 26762461

View details for PubMedCentralID PMC4843164
Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex. Structure (London, England : 1993) Cheng, S., Seven, A. B., Wang, J., Skiniotis, G., �zkan, E. 2016; 24 (12): 2163?73
Abstract

Synaptic specificity is a defining property of neural networks. In the cerebellum, synapses between parallel fiber neurons and Purkinje cells are specified by the simultaneous interactions of secreted protein cerebellin with pre-synaptic neurexin and post-synaptic delta-type glutamate receptors (GluD). Here, we determined the crystal structures of the trimeric C1q-like domain of rat cerebellin-1, and the first complete ectodomain of a GluD, rat GluD2. Cerebellin binds to the LNS6 domain of ?- and ?-neurexin-1 through a high-affinity interaction that involves its highly flexible N-terminal domain. In contrast, we show that the interaction of cerebellin with isolated GluD2 ectodomain is low affinity, which is not simply an outcome of lost avidity when compared with binding with a tetrameric full-length receptor. Rather, high-affinity capture of cerebellin by post-synaptic terminals is likely controlled by long-distance regulation within this transsynaptic complex. Altogether, our results suggest unusual conformational flexibility within all components of the complex.

View details for DOI 10.1016/j.str.2016.11.004

View details for PubMedID 27926833

View details for PubMedCentralID PMC5149402
Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly PLOS PATHOGENS Xu, K., Chan, Y., Bradel-Tretheway, B., Akyol-Ataman, Z., Zhu, Y., Dutta, S., Yan, L., Feng, Y., Wang, L., Skiniotis, G., Lee, B., Zhou, Z. H., Broder, C. C., Aguilar, H. C., Nikolov, D. B. 2015; 11 (12)
Abstract

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

View details for DOI 10.1371/journal.ppat.1005322

View details for Web of Science ID 000368332800040

View details for PubMedID 26646856

View details for PubMedCentralID PMC4672880
Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds PLOS ONE Verma, V., Mallik, L., Hariadi, R. F., Sivaramakrishnan, S., Skiniotis, G., Joglekar, A. P. 2015; 10 (9)
Abstract

DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

View details for DOI 10.1371/journal.pone.0137125

View details for Web of Science ID 000360897600022

View details for PubMedID 26348722

View details for PubMedCentralID PMC4562706
ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation JOURNAL OF VIROLOGY Inoue, T., Dosey, A., Herbstman, J. F., Ravindran, M. S., Skiniotis, G., Tsai, B. 2015; 89 (17): 8897-8908
Abstract

The nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process.PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies for preventing and treating PyV-related diseases.

View details for DOI 10.1128/JVI.00941-15

View details for Web of Science ID 000360703900021

View details for PubMedID 26085143

View details for PubMedCentralID PMC4524074
Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII BLOOD Yee, A., Oleskie, A. N., Dosey, A. M., Kretz, C. A., Gildersleeve, R. D., Dutta, S., Su, M., Ginsburg, D., Skiniotis, G. 2015; 126 (8): 939-942
Abstract

Binding to the von Willebrand factor (VWF) D'D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D'D3 alone and in complex with FVIII. The D'D3 dimer ([D'D3]2) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D'. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D' domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. Modeling the FVIII structure into the three-dimensional EM reconstructions of [D'D3]2-FVIII ternary and quaternary complexes indicates conformational rearrangements of the FVIII C domains compared with their disposition in the unbound state. These results illustrate the cooperative plasticity between VWF and FVIII that coordinate their high-affinity interaction.

View details for DOI 10.1182/blood-2015-04-641696

View details for Web of Science ID 000360535700011

View details for PubMedID 26065653

View details for PubMedCentralID PMC4543228
Electron Microscopic Visualization of Protein Assemblies on Flattened DNA Origami ACS NANO Mallik, L., Dhakal, S., Nichols, J., Mahoney, J., Dosey, A. M., Jiang, S., Sunahara, R. K., Skiniotis, G., Walter, N. G. 2015; 9 (7): 7133-7141
Abstract

DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson-Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ?2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications.

View details for DOI 10.1021/acsnano.5b01841

View details for Web of Science ID 000358823200050

View details for PubMedID 26149412
Architecture of the polyketide synthase module: surprises from electron cryo-microscopy CURRENT OPINION IN STRUCTURAL BIOLOGY Smith, J. L., Skiniotis, G., Sherman, D. H. 2015; 31: 9-19
Abstract

Modular polyketide synthases (PKS) produce a vast array of bioactive molecules that are the basis of many highly valued pharmaceuticals. The biosynthesis of these compounds is based on ordered assembly lines of multi-domain modules, each extending and modifying a specific chain-elongation intermediate before transfer to the next module for further processing. The first 3D structures of a full polyketide synthase module in different functional states were obtained recently by electron cryo-microscopy. The unexpected module architecture revealed a striking evolutionary divergence of the polyketide synthase compared to its metazoan fatty acid synthase homolog, as well as remarkable conformational rearrangements dependent on its biochemical state during the full catalytic cycle. The design and dynamics of the module are highly optimized for both catalysis and fidelity in the construction of complex, biologically active natural products.

View details for DOI 10.1016/j.sbi.2015.02.014

View details for Web of Science ID 000357706000004

View details for PubMedID 25791608

View details for PubMedCentralID PMC4476912
A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation GENES & DEVELOPMENT Zhang, P., Chaturvedi, C., Tremblay, V., Cramet, M., Brunzelle, J. S., Skiniotis, G., Brand, M., Shilatifard, A., Couture, J. 2015; 29 (2): 123-128
Abstract

The methyltransferase activity of the trithorax group (TrxG) protein MLL1 found within its COMPASS (complex associated with SET1)-like complex is allosterically regulated by a four-subunit complex composed of WDR5, RbBP5, Ash2L, and DPY30 (also referred to as WRAD). We report structural evidence showing that in WRAD, a concave surface of the Ash2L SPIa and ryanodine receptor (SPRY) domain binds to a cluster of acidic residues, referred to as the D/E box, in RbBP5. Mutational analysis shows that residues forming the Ash2L/RbBP5 interface are important for heterodimer formation, stimulation of MLL1 catalytic activity, and erythroid cell terminal differentiation. We also demonstrate that a phosphorylation switch on RbBP5 stimulates WRAD complex formation and significantly increases KMT2 (lysine [K] methyltransferase 2) enzyme methylation rates. Overall, our findings provide structural insights into the assembly of the WRAD complex and point to a novel regulatory mechanism controlling the activity of the KMT2/COMPASS family of lysine methyltransferases.

View details for DOI 10.1101/gad.254870.114

View details for Web of Science ID 000347969500002

View details for PubMedID 25593305

View details for PubMedCentralID PMC4298132
2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy. Methods in molecular biology (Clifton, N.J.) Peisley, A., Skiniotis, G. 2015; 1335: 29-38
Abstract

While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (<150 kDa) complexes between GPCRs and their partner proteins. Negative stain EM, whereby the biological samples are embedded in a thin layer of heavy metal solution, is a well-established alternative technique that provides the enhanced contrast needed to visualize small macromolecular complexes. This methodology can offer a simple and powerful tool for the rapid evaluation of sample characteristics, such as homogeneity or oligomeric state. When coupled to single particle classification and averaging, negative stain EM can provide valuable information on the overall architecture and dynamics of protein complexes. Here we provide a concise protocol for negative stain imaging and two-dimensional (2D) projection analysis of GPCR complexes, including notes for the intricacies of the application in these biological systems.

View details for DOI 10.1007/978-1-4939-2914-6_3

View details for PubMedID 26260592
Molecular Basis for DPY-30 Association to COMPASS-like and NURF Complexes STRUCTURE Tremblay, V., Zhang, P., Chaturvedi, C., Thornton, J., Brunzelle, J. S., Skiniotis, G., Shilatifard, A., Brand, M., Couture, J. 2014; 22 (12): 1821-1830
Abstract

DPY-30 is a subunit of mammalian COMPASS-like complexes (complex of proteins associated with Set1) and regulates global histone H3 Lys-4 trimethylation. Here we report structural evidence showing that the incorporation of DPY-30 into COMPASS-like complexes is mediated by several hydrophobic interactions between an amphipathic ? helix located on the C terminus of COMPASS subunit ASH2L and the inner surface of the DPY-30 dimerization/docking (D/D) module. Mutations impairing the interaction between ASH2L and DPY-30 result in a loss of histone H3K4me3 at the ? locus control region and cause a delay in erythroid cell terminal differentiation. Using overlay assays, we defined a consensus sequence for DPY-30 binding proteins and found that DPY-30 interacts with BAP18, a subunit of the nucleosome remodeling factor complex. Overall, our results indicate that the ASH2L/DPY-30 complex is important for cell differentiation and provide insights into the ability of DPY-30 to associate with functionally divergent multisubunit complexes.

View details for DOI 10.1016/j.str.2014.10.002

View details for Web of Science ID 000345898700015

View details for PubMedID 25456412

View details for PubMedCentralID PMC5832440
Collective Variable Approaches for Single Molecule Flexible Fitting and Enhanced Sampling CHEMICAL REVIEWS Vashisth, H., Skiniotis, G., Brooks, C. L. 2014; 114 (6): 3353-3365
View details for DOI 10.1021/cr4005988

View details for Web of Science ID 000333552000012

View details for PubMedID 24446720

View details for PubMedCentralID PMC3983124
Flavivirus NS1 Structures Reveal Surfaces for Associations with Membranes and the Immune System SCIENCE Akey, D. L., Brown, W. C., Dutta, S., Konwerski, J., Jose, J., Jurkiw, T. J., DelProposto, J., Ogata, C. M., Skiniotis, G., Kuhn, R. J., Smith, J. L. 2014; 343 (6173): 881-885
Abstract

Flaviviruses, the human pathogens responsible for dengue fever, West Nile fever, tick-borne encephalitis, and yellow fever, are endemic in tropical and temperate parts of the world. The flavivirus nonstructural protein 1 (NS1) functions in genome replication as an intracellular dimer and in immune system evasion as a secreted hexamer. We report crystal structures for full-length, glycosylated NS1 from West Nile and dengue viruses. The NS1 hexamer in crystal structures is similar to a solution hexamer visualized by single-particle electron microscopy. Recombinant NS1 binds to lipid bilayers and remodels large liposomes into lipoprotein nanoparticles. The NS1 structures reveal distinct domains for membrane association of the dimer and interactions with the immune system and are a basis for elucidating the molecular mechanism of NS1 function.

View details for DOI 10.1126/science.1247749

View details for Web of Science ID 000331552600045

View details for PubMedID 24505133

View details for PubMedCentralID PMC4263348
Characterization of a highly flexible self-assembling protein system designed to form nanocages PROTEIN SCIENCE Patterson, D. P., Su, M., Franzmann, T. M., Sciore, A., Skiniotis, G., Marsh, E. N. 2014; 23 (2): 190-199
Abstract

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(-), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C3 symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(-) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(-) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.

View details for DOI 10.1002/pro.2405

View details for Web of Science ID 000329939900006

View details for PubMedID 24318954

View details for PubMedCentralID PMC3926744
Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation GENES & DEVELOPMENT Thornton, J. L., Westfield, G. H., Takahashi, Y., Cook, M., Gao, X., Woodfin, A. R., Lee, J., Morgan, M. A., Jackson, J., Smith, E. R., Couture, J., Skiniotis, G., Shilatifard, A. 2014; 28 (2): 115-120
Abstract

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner.

View details for DOI 10.1101/gad.232215.113

View details for Web of Science ID 000330572300002

View details for PubMedID 24402317

View details for PubMedCentralID PMC3909785
Inhibition of AMPK Catabolic Action by GSK3 MOLECULAR CELL Suzuki, T., Bridges, D., Nakada, D., Skiniotis, G., Morrison, S. J., Lin, J. D., Saltiel, A. R., Inoki, K. 2013; 50 (3): 407-419
Abstract

AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK ?�regulatory subunit and phosphorylates the AMPK ? catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the ? subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK.

View details for DOI 10.1016/j.molcel.2013.03.022

View details for Web of Science ID 000319183500011

View details for PubMedID 23623684

View details for PubMedCentralID PMC3654099
Enhanced Sampling and Overfitting Analyses in Structural Refinement of Nucleic Acids into Electron Microscopy Maps JOURNAL OF PHYSICAL CHEMISTRY B Vashisth, H., Skiniotis, G., Brooks, C. L. 2013; 117 (14): 3738-3746
Abstract

Flexible fitting computational algorithms are often useful to interpret low-resolution maps of many macromolecular complexes generated by electron microscopy (EM) imaging. One such atomistic simulation technique is molecular dynamics flexible fitting (MDFF), which has been widely applied to generate structural models of large ribonucleoprotein assemblies such as the ribosome. We have previously shown that MDFF simulations of globular proteins are sensitive to the resolution of the target EM map and the strength of restraints used to preserve the secondary structure elements during fitting (Vashisth, H.; et al. Structure 2012, 20, 1453-1462). In this work, we aim to systematically examine the quality of structural models of various nucleic acids obtained via MDFF by varying the map resolution and the strength of structural restraints. We also demonstrate how an enhanced conformational sampling technique for proteins, temperature-accelerated molecular dynamics (TAMD), can be combined with MDFF for the structural refinement of nucleic acids in EM maps. Finally, we also demonstrate application of TAMD-assisted MDFF (TAMDFF) on a RNA/protein complex and suggest that TAMDFF is a viable strategy for enhanced conformational fitting in target maps of ribonucleoprotein complexes.

View details for DOI 10.1021/jp3126297

View details for Web of Science ID 000317552700005

View details for PubMedID 23506287

View details for PubMedCentralID PMC3690198
Assembling a COMPASS EPIGENETICS Couture, J., Skiniotis, G. 2013; 8 (4): 349-354
Abstract

Post-translational modifications of histone proteins lie at the heart of the epigenetic landscape in the cell's nucleus and the precise regulation of gene expression. A myriad of studies have showed that several histone-modifying enzymes are controlled by modulatory protein partner subunits and other post-transcriptional modifications deposited in the vicinity of the targeted site. All together, these mechanisms create an intricate network of interactions that regulate enzymatic activities and ultimately control the site-specific deposition of covalent modifications. In this Point-of-View, we discuss our evolving understanding on the assembly and architecture of histone H3 Lys-4 (H3K4) methyltransferase COMPASS complexes and the techniques that progressively allowed us to better define the molecular basis of complex formation and function. We further briefly discuss some of the challenges lying ahead and additional approaches required to understand mechanistic details for the function of such complexes.

View details for DOI 10.4161/epi.24177

View details for Web of Science ID 000323176600002

View details for PubMedID 23470558

View details for PubMedCentralID PMC3674043
Full-length G alpha(q)-phospholipase C-beta 3 structure reveals interfaces of the C-terminal coiled-coil domain NATURE STRUCTURAL & MOLECULAR BIOLOGY Lyon, A. M., Dutta, S., Boguth, C. A., Skiniotis, G., Tesmer, J. J. 2013; 20 (3): 355-362
Abstract

Phospholipase C-? (PLC?) is directly activated by G?q, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLC?3 in complex with mouse G?q. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of G?q in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

View details for DOI 10.1038/nsmb.2497

View details for Web of Science ID 000316041000018

View details for PubMedID 23377541

View details for PubMedCentralID PMC3594540
Paclitaxel-Conjugated PAMAM Dendrimers Adversely Affect Microtubule Structure through Two Independent Modes of Action BIOMACROMOLECULES Cline, E. N., Li, M., Choi, S. K., Herbstman, J. F., Kaul, N., Meyhoefer, E., Skiniotis, G., Baker, J. R., Larson, R. G., Walter, N. G. 2013; 14 (3): 654-664
Abstract

Paclitaxel (Taxol) is an anticancer drug that induces mitotic arrest via microtubule hyperstabilization but causes side effects due to its hydrophobicity and cellular promiscuity. The targeted cytotoxicity of hydrophilic paclitaxel-conjugated polyamidoamine (PAMAM) dendrimers has been demonstrated in cultured cancer cells. Mechanisms of action responsible for this cytotoxicity are unknown, that is, whether the cytotoxicity is due to paclitaxel stabilization of microtubules, as is whether paclitaxel is released intracellularly from the dendrimer. To determine whether the conjugated paclitaxel can bind microtubules, we used a combination of ensemble and single microtubule imaging techniques in vitro. We demonstrate that these conjugates adversely affect microtubules by (1) promoting the polymerization and stabilization of microtubules in a paclitaxel-dependent manner, and (2) bundling preformed microtubules in a paclitaxel-independent manner, potentially due to protonation of tertiary amines in the dendrimer interior. Our results provide mechanistic insights into the cytotoxicity of paclitaxel-conjugated PAMAM dendrimers and uncover unexpected risks of using such conjugates therapeutically.

View details for DOI 10.1021/bm301719b

View details for Web of Science ID 000316044700009

View details for PubMedID 23391096

View details for PubMedCentralID PMC3603340
Phleboviruses encapsidate their genomes by sequestering RNA bases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Raymond, D. D., Piper, M. E., Gerrard, S. R., Skiniotis, G., Smith, J. L. 2012; 109 (47): 19208-19213
Abstract

Rift Valley fever and Toscana viruses are human pathogens for which no effective therapeutics exist. These and other phleboviruses have segmented negative-sense RNA genomes that are sequestered by a nucleocapsid protein (N) to form ribonucleoprotein (RNP) complexes of irregular, asymmetric structure, previously uncharacterized at high resolution. N binds nonspecifically to single-stranded RNA with nanomolar affinity. Crystal structures of Rift Valley fever virus N-RNA complexes reconstituted with defined RNAs of different length capture tetrameric, pentameric and hexameric N-RNA multimers. All N-N subunit contacts are mediated by a highly flexible ?-helical arm. Arm movement gives rise to the three multimers in the crystal structures and also explains the asymmetric architecture of the RNP. Despite the flexible association of subunits, the crystal structures reveal an invariant, monomeric RNP building block, consisting of the core of one N subunit, the arm of a neighboring N, and four RNA nucleotides with the flanking phosphates. Up to three additional RNA nucleotides bind between subunits. The monomeric building block is matched in size to the repeating unit in viral RNP, as visualized by electron microscopy. N sequesters four RNA bases in a narrow hydrophobic binding slot and has polar contacts only with the sugar-phosphate backbone, which faces the solvent. All RNA bases, whether in the binding slot or in the subunit interface, face the protein in a manner that is incompatible with base pairing or with "reading" by the viral polymerase.

View details for DOI 10.1073/pnas.1213553109

View details for Web of Science ID 000311997200044

View details for PubMedID 23129612

View details for PubMedCentralID PMC3511139
Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins JOURNAL OF VIROLOGY Chan, Y., Lu, M., Dutta, S., Yan, L., Barr, J., Flora, M., Feng, Y., Xu, K., Nikolov, D. B., Wang, L., Skiniotis, G., Broder, C. C. 2012; 86 (21): 11457-11471
Abstract

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF(GCNt)), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F(0) precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF(GCNt)) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF(GCNt) constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF(GCNt) and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.

View details for DOI 10.1128/JVI.01318-12

View details for Web of Science ID 000309657100005

View details for PubMedID 22915804

View details for PubMedCentralID PMC3486283
Luminal Localization of alpha-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules PLOS ONE Soppina, V., Herbstman, J. F., Skiniotis, G., Verhey, K. J. 2012; 7 (10)
Abstract

The ??-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of ?-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

View details for DOI 10.1371/journal.pone.0048204

View details for Web of Science ID 000310262500051

View details for PubMedID 23110214

View details for PubMedCentralID PMC3482196
Using Enhanced Sampling and Structural Restraints to Refine Atomic Structures into Low-Resolution Electron Microscopy Maps STRUCTURE Vashisth, H., Skiniotis, G., Brooks, C. L. 2012; 20 (9): 1453-1462
Abstract

For a variety of problems in structural biology, low-resolution maps generated by electron microscopy imaging are often interpreted with the help of various flexible-fitting computational algorithms. In this work, we systematically analyze the quality of final models of various proteins obtained via molecular dynamics flexible fitting (MDFF) by varying the map-resolution, strength of structural restraints, and the steering forces. We find that MDFF can be extended to understand conformational changes in lower-resolution maps if larger structural restraints and lower steering forces are used to prevent overfitting. We further show that the capabilities of MDFF can be extended by combining it with an enhanced conformational sampling method, temperature-accelerated molecular dynamics (TAMD). Specifically, either TAMD can be used to generate better starting configurations for MDFF fitting or TAMD-assisted MDFF (TAMDFF) can be performed to accelerate conformational search in atomistic simulations.

View details for DOI 10.1016/j.str.2012.08.007

View details for Web of Science ID 000308682700004

View details for PubMedID 22958641

View details for PubMedCentralID PMC3438525
Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK) CELL STRESS & CHAPERONES Thompson, A. D., Bernard, S. M., Skiniotis, G., Gestwicki, J. E. 2012; 17 (3): 313-327
Abstract

The molecular chaperone DnaK binds to exposed hydrophobic segments in proteins, protecting them from aggregation. DnaK interacts with protein substrates via its substrate-binding domain, and the affinity of this interaction is allosterically regulated by its nucleotide-binding domain. In addition to regulating interdomain allostery, the nucleotide state has been found to influence homo-oligomerization of DnaK. However, the architecture of oligomeric DnaK and its potential functional relevance in the chaperone cycle remain undefined. Towards that goal, we examined the structures of DnaK by negative stain electron microscopy. We found that DnaK samples contain an ensemble of monomers, dimers, and other small, defined multimers. To better understand the function of these oligomers, we stabilized them by cross-linking and found that they retained ATPase activity and protected a model substrate from denaturation. However, these oligomers had a greatly reduced ability to refold substrate and did not respond to stimulation by DnaJ. Finally, we observed oligomeric DnaK in Escherichia coli cellular lysates by native gel electrophoresis and found that these structures became noticeably more prevalent in cells exposed to heat shock. Together, these studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling.

View details for DOI 10.1007/s12192-011-0307-1

View details for Web of Science ID 000302227200004

View details for PubMedID 22076723

View details for PubMedCentralID PMC3312962
Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Takahashi, Y., Westfield, G. H., Oleskie, A. N., Trievel, R. C., Shilatifard, A., Skiniotis, G. 2011; 108 (51): 20526-20531
Abstract

Histone H3 lysine 4 (H3K4) methylation is catalyzed by the highly evolutionarily conserved multiprotein complex known as Set1/COMPASS or MLL/COMPASS-like complexes from yeast to human, respectively. Here we have reconstituted fully functional yeast Set1/COMPASS and human MLL/COMPASS-like complex in vitro and have identified the minimum subunit composition required for histone H3K4 methylation. These subunits include the methyltransferase C-terminal SET domain of Set1/MLL, Cps60/Ash2L, Cps50/RbBP5, Cps30/WDR5, and Cps25/Dpy30, which are all common components of the COMPASS family from yeast to human. Three-dimensional (3D) cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and two-dimensional (2D) EM analysis of the individual subcomplexes reveal a Y-shaped architecture with Cps50 and Cps30 localizing on the top two adjacent lobes and Cps60-Cps25 forming the base at the bottom. EM analysis of the human complex reveals a striking similarity to its yeast counterpart, suggesting a common subunit organization. The SET domain of Set1 is located at the juncture of Cps50, Cps30, and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation. This structural arrangement suggested that COMPASS family members function as exo-methylases, which we have confirmed by in vitro and in vivo studies.

View details for DOI 10.1073/pnas.1109360108

View details for Web of Science ID 000298289400057

View details for PubMedID 22158900

View details for PubMedCentralID PMC3251153
Structural Snapshots of Full-Length Jak1, a Transmembrane gp130/IL-6/IL-6R alpha Cytokine Receptor Complex, and the Receptor-Jak1 Holocomplex STRUCTURE Lupardus, P. J., Skiniotis, G., Rice, A. J., Thomas, C., Fischer, S., Walz, T., Garcia, K. C. 2011; 19 (1): 45-55
Abstract

The shared cytokine receptor gp130 signals as�a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here, we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6R?) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to form a continuous connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three lobed structure comprising FERM-SH2, pseudokinase, and kinase modules possessing extensive intersegmental flexibility that likely facilitates allosteric activation. Single-particle imaging of�the gp130/IL-6/IL-6R?/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of�a membrane environment.

View details for DOI 10.1016/j.str.2010.10.010

View details for PubMedID 21220115
Tandem Acyl Carrier Proteins in the Curacin Biosynthetic Pathway Promote Consecutive Multienzyme Reactions with a Synergistic Effect ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Gu, L., Eisman, E. B., Dutta, S., Franzmann, T. M., Walter, S., Gerwick, W. H., Skiniotis, G., Sherman, D. H. 2011; 50 (12): 2795-2798
View details for DOI 10.1002/anie.201005280

View details for Web of Science ID 000288173400026

View details for PubMedID 21387490

View details for PubMedCentralID PMC3081611
Structural basis of Dscam isoform specificity NATURE Meijers, R., Puettmann-Holgado, R., Skiniotis, G., Liu, J., Walz, T., Wang, J., Schmucker, D. 2007; 449 (7161): 487-U12
Abstract

The Dscam gene gives rise to thousands of diverse cell surface receptors thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

View details for DOI 10.1038/nature06147

View details for Web of Science ID 000249724800046

View details for PubMedID 17721508
Acetylated histone tail peptides induce structural rearrangements in the RSC chromatin remodeling complex JOURNAL OF BIOLOGICAL CHEMISTRY Skiniotis, G., Moazed, D., Walz, T. 2007; 282 (29): 20804-20808
Abstract

Post-translational acetylation of histone tails is often required for the recruitment of ATP-dependent chromatin remodelers, which in turn mobilize nucleosomes on the chromatin fiber. Here we show that the lower lobe of the ATP-dependent chromatin remodeler RSC exists in a dynamic equilibrium and can be found extended away or retracted against the tripartite upper lobe of the complex. Extension of the lower lobe increases the size of a central cavity that has been proposed to be the nucleosome binding site. We show that the presence of acetylated histone 3 N-terminal tail peptides stabilizes the lower lobe of RSC in the retracted state, suggesting that domains recognizing the acetylated histone tails reside at the interface between the two lobes. Based on three-dimensional reconstructions, we propose a model for the interaction of RSC with acetylated nucleosomes.

View details for DOI 10.1074/jbc.C700081200

View details for Web of Science ID 000248047500005

View details for PubMedID 17535815
Surface-decoration of microtubules by human Tau JOURNAL OF MOLECULAR BIOLOGY Santarella, R. A., Skiniotis, G., Goldie, K. N., Tittmann, P., Gross, H., Mandelkow, E. M., Mandelkow, E., Hoenger, A. 2004; 339 (3): 539-553
Abstract

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a "halo" of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on alpha-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.

View details for DOI 10.1016/j.jmb.2004.04.008

View details for Web of Science ID 000221773200006

View details for PubMedID 15147841
Modulation of kinesin binding by the C-termini of tubulin EMBO JOURNAL Skiniotis, G., Cochran, J. C., Muller, J., Mandelkow, E., Gilbert, S. P., Hoenger, A. 2004; 23 (5): 989-999
Abstract

The flexible tubulin C-terminal tails (CTTs) have recently been implicated in the walking mechanism of dynein and kinesin. To address their role in the case of conventional kinesin, we examined the structure of kinesin-microtubule (MT) complexes before and after CTT cleavage by subtilisin. Our results show that the CTTs directly modulate the motor-tubulin interface and the binding properties of motors. CTT cleavage increases motor binding stability, and kinesin appears to adopt a binding conformation close to the nucleotide-free configuration under most nucleotide conditions. Moreover, C-terminal cleavage results in trapping a transient motor-ADP-MT intermediate. Using SH3-tagged dimeric and monomeric constructs, we could also show that the position of the kinesin neck is not affected by the C-terminal segments of tubulin. Overall, our study reveals that the tubulin C-termini define the stability of the MT-kinesin complex in a nucleotide-dependent manner, and highlights the involvement of tubulin in the regulation of weak and strong kinesin binding states.

View details for DOI 10.1038/sj.emboj.7600118

View details for Web of Science ID 000220694200001

View details for PubMedID 14976555

View details for PubMedCentralID PMC380974
A kinesin-like motor inhibits microtubule dynamic instability SCIENCE Bringmann, H., Skiniotis, G., Spilker, A., Kandels-Lewis, S., Vernos, I., Surrey, T. 2004; 303 (5663): 1519-1522
Abstract

The motility of molecular motors and the dynamic instability of microtubules are key dynamic processes for mitotic spindle assembly and function. We report here that one of the mitotic kinesins that localizes to chromosomes, Xklp1 from Xenopus laevis, could inhibit microtubule growth and shrinkage. This effect appeared to be mediated by a structural change in the microtubule lattice. We also found that Xklp1 could act as a fast, nonprocessive, plus end-directed molecular motor. The integration of the two properties, motility and inhibition of microtubule dynamics, in one molecule emphasizes the versatile properties of kinesin family members.

View details for Web of Science ID 000220000100043

View details for PubMedID 15001780
Nucleotide-induced conformations in the neck region of dimeric kinesin EMBO JOURNAL Skiniotis, G., Surrey, T., Altmann, S., Gross, H., Song, Y. H., Mandelkow, E., Hoenger, A. 2003; 22 (7): 1518-1528
Abstract

The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP-PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration.

View details for Web of Science ID 000182159500008

View details for PubMedID 12660159

View details for PubMedCentralID PMC152908
Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd EMBO JOURNAL Wendt, T. G., Volkmann, N., Skiniotis, G., Goldie, K. N., Muller, J., Mandelkow, E., Hoenger, A. 2002; 21 (22): 5969-5978
Abstract

We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction.

View details for Web of Science ID 000179446900003

View details for PubMedID 12426369

View details for PubMedCentralID PMC137211
Identification of medically significant fungal genera by polymerase chain reaction followed by restriction enzyme analysis FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY Velegraki, A., Kambouris, M. E., Skiniotis, G., Savala, M., Mitroussia-Ziouva, A., Legakis, N. J. 1999; 23 (4): 303-312
Abstract

Rapid non-culture-dependent assays for identification of fungi quicken diagnosis and prompt treatment of invasive fungal disease. Fungal DNA extracts from pure cultures of the most frequently isolated fungal pathogens belonging to the Genera Aspergillus, Candida and Cryptococcus along with less common pathogenic Genera were amplified with the general fungal primer pair internal transcribed spacer-1/4. Subsequently, the amplicon was digested with the restriction endonucleases MspI, HaeIII, HinfI and EcoRI in order to generate genus- or species-specific patterns for identification of the fungus. HinfI produced indistinguishable fingerprints for all Aspergillus species tested. MspI produced species-specific patterns for: Cryptococcus neoformans, Cryptococcus non-neoformans, Candida albicans and Candida tropicalis. EcoRI succeeded in differentiating penicillia from aspergilli and cryptococci from Candida spp. It is concluded that this procedure can differentiate genera and occasionally species of medically important fungi and that following the necessary validation experiments, it can be used directly on clinical samples to assist prompt diagnosis of systemic fungal infections.

View details for Web of Science ID 000079785200005

View details for PubMedID 10225290

Georgios Skiniotis

Professor of Molecular and Cellular Physiology, of Structural Biology and of Photon Science

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