Doctor of Philosophy, Scripps Research Institute Kellogg School (2014)
Michael Snyder, Postdoctoral Faculty Sponsor
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR?) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPAR? by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently, we reported the development of PPAR? antagonist SR1664, designed to block the obesity-induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin-sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPAR?, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow-derived mesenchymal stem cells with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand-mediated PPAR? repression, and suggest a therapeutic approach to promote bone formation.
View details for DOI 10.1038/ncomms8443
View details for PubMedID 26068133
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS or DXMS) has emerged as an important tool for the development of small molecule therapeutics and biopharmaceuticals. Central to these advances have been improvements to automated HDX-MS platforms and software that allow for the rapid acquisition and processing of experimental data. Correlating the HDX-MS profile of large numbers of ligands with their functional outputs has enabled the development of structure activity relationships (SAR) and delineation of ligand classes based on functional selectivity. HDX-MS has also been applied to address many of the unique challenges posed by the continued emergence of biopharmaceuticals. Here we review the latest applications of HDX-MS to drug discovery, recent advances in technology and software, and provide perspective on future outlook.
View details for DOI 10.1016/j.sbi.2014.08.007
View details for Web of Science ID 000345203000015
View details for PubMedID 25179005
PPAR? is a target for insulin-sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPAR?. Here we reveal that synthetic PPAR? ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPAR? hyperactivation in vivo, perhaps explaining why PPAR? full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPAR? activation by ligands and suggest that allosteric modulators could be designed to fine tune PPAR? activity without competing with endogenous ligands.
View details for DOI 10.1038/ncomms4571
View details for Web of Science ID 000335220300007
View details for PubMedID 24705063
Nuclear receptors (NRs) play central roles in metabolic syndrome, making them attractive drug targets despite the challenge of achieving functional selectivity. For instance, members of the thiazolidinedione class of insulin sensitizers offer robust efficacy but have been limited due to adverse effects linked to activation of genes not involved in insulin sensitization. Studies reviewed here provide strategies for targeting subsets of PPAR? target genes, enabling development of next-generation modulators with improved therapeutic index. Additionally, emerging evidence suggests that targeting the NRs ROR and Rev-erb holds promise for treating metabolic syndrome based on their involvement in circadian rhythm and metabolism.
View details for DOI 10.1016/j.cmet.2013.12.009
View details for Web of Science ID 000330722600006
View details for PubMedID 24440037
PPAR? is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPAR?-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPAR? by Cdk5. Here we describe novel synthetic compounds that have a unique mode of binding to PPAR?, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPAR? drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPAR?.
View details for DOI 10.1038/nature10383
View details for Web of Science ID 000295080500043
View details for PubMedID 21892191
Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 ┼ resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 ┼ over 93% of the NGO1391 protein), despite an extremely low sequence identity of ?15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ?200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.
View details for DOI 10.1002/pro.497
View details for Web of Science ID 000283759000010
View details for PubMedID 20836087
View details for PubMedCentralID PMC3005784
Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (?40-99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.
View details for DOI 10.1107/S1744309109022672
View details for Web of Science ID 000282503300007
View details for PubMedID 20944208
View details for PubMedCentralID PMC2954202
A novel aminoacyl-tRNA synthetase that contains an iron-sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 18.104.22.168) reveals an iron-sulfur [4Fe-4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an L-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe-4S] cluster-binding motif (C-x??-C-x?-C-x?-C). It is speculated that the iron-sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.
View details for DOI 10.1107/S1744309110037619
View details for Web of Science ID 000282503300028
View details for PubMedID 20944229
View details for PubMedCentralID PMC2954223
Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.
View details for DOI 10.1107/S1744309110023055
View details for Web of Science ID 000282503300024
View details for PubMedID 20944225
View details for PubMedCentralID PMC2954219
The crystal structure of PA1994 from Pseudomonas aeruginosa, a member of the Pfam PF06475 family classified as a domain of unknown function (DUF1089), reveals a novel fold comprising a 15-stranded ?-sheet wrapped around a single ?-helix that assembles into a tight dimeric arrangement. The remote structural similarity to lipoprotein localization factors, in addition to the presence of an acidic pocket that is conserved in DUF1089 homologs, phospholipid-binding and sugar-binding proteins, indicate a role for PA1994 and the DUF1089 family in glycolipid metabolism. Genome-context analysis lends further support to the involvement of this family of proteins in glycolipid metabolism and indicates possible activation of DUF1089 homologs under conditions of bacterial cell-wall stress or host-pathogen interactions.
View details for DOI 10.1107/S1744309109022684
View details for Web of Science ID 000282503300012
View details for PubMedID 20944213
View details for PubMedCentralID PMC2954207
The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01?┼ using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.
View details for DOI 10.1107/S1744309110007517
View details for Web of Science ID 000282503300006
View details for PubMedID 20944207
View details for PubMedCentralID PMC2954201
Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 22.214.171.124), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like ?+? core domain (NTD) consisting of a five-stranded, mixed ?-sheet flanked by several ?-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.
View details for DOI 10.1107/S1744309110020166
View details for Web of Science ID 000282503300029
View details for PubMedID 20944230
View details for PubMedCentralID PMC2954224
The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively.
View details for DOI 10.1107/S1744309109037749
View details for Web of Science ID 000282503300004
View details for PubMedID 20944205
View details for PubMedCentralID PMC2954199
In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6?┼ resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006), Proteins, 65, 1046-1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein.
View details for DOI 10.1107/S1744309109021988
View details for Web of Science ID 000282503300030
View details for PubMedID 20944231
View details for PubMedCentralID PMC2954225
Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a ?-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to ?-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.
View details for DOI 10.1107/S1744309109046788
View details for Web of Science ID 000282503300020
View details for PubMedID 20944221
View details for PubMedCentralID PMC2954215
The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7?┼ and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.
View details for DOI 10.1107/S1744309110032999
View details for Web of Science ID 000282503300021
View details for PubMedID 20944222
View details for PubMedCentralID PMC2954216
Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific ?-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8?┼ resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-?-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and ?-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-?-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.
View details for DOI 10.1107/S1744309110021214
View details for Web of Science ID 000282503300031
View details for PubMedID 20944232
View details for PubMedCentralID PMC2954226
The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel ?+? fold comprising two ?-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.
View details for DOI 10.1107/S1744309109023689
View details for Web of Science ID 000282503300011
View details for PubMedID 20944212
View details for PubMedCentralID PMC2954206
SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein-protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).
View details for DOI 10.1107/S1744309110002514
View details for Web of Science ID 000282503300005
View details for PubMedID 20944206
View details for PubMedCentralID PMC2954200
Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)(+)-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD(+) has been determined to 2.0?┼ resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal ?/? dinucleotide-binding domain and a C-terminal ?-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an ?-? motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP(+) and NAD(+). The loop between ?5 and ?6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between ?5 and ?6) and Arg365' (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.
View details for DOI 10.1107/S1744309110021688
View details for Web of Science ID 000282503300027
View details for PubMedID 20944228
View details for PubMedCentralID PMC2954222
KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.
View details for DOI 10.1107/S1744309109018168
View details for Web of Science ID 000282503300018
View details for PubMedID 20944219
View details for PubMedCentralID PMC2954213
BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.
View details for DOI 10.1107/S1744309110006548
View details for Web of Science ID 000282503300022
View details for PubMedID 20944223
View details for PubMedCentralID PMC2954217
The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.
View details for DOI 10.1107/S1744309109050416
View details for Web of Science ID 000282503300013
View details for PubMedID 20944214
View details for PubMedCentralID PMC2954208
The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1?┼ by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.
View details for DOI 10.1107/S1744309109050647
View details for Web of Science ID 000282503300008
View details for PubMedID 20944209
View details for PubMedCentralID PMC2954203
The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25?┼ resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the ?/? SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their ?2 and ?3 helices. In the `open' conformation (YP_001095227.1), these helices are 15?┼ apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, ?2 and ?3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.
View details for DOI 10.1107/S1744309109042481
View details for Web of Science ID 000282503300017
View details for PubMedID 20944218
View details for PubMedCentralID PMC2954212
The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member ofáthe Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45?┼ by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.
View details for DOI 10.1107/S1744309109025196
View details for Web of Science ID 000282503300010
View details for PubMedID 20944211
View details for PubMedCentralID PMC2954205
The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78?┼ resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-?-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other ?-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.
View details for DOI 10.1107/S1744309110025534
View details for Web of Science ID 000282503300016
View details for PubMedID 20944217
View details for PubMedCentralID PMC2954211
BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05?┼ resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a ?-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a ?-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal ?-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.
View details for DOI 10.1107/S1744309110028228
View details for Web of Science ID 000282503300023
View details for PubMedID 20944224
View details for PubMedCentralID PMC2954218
Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks. As X-ray structural information has been available only for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is an Mre11 endo/exonuclease (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only approximately 20%. However, they differ substantially in their DNA-specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA-specificity domain are not. The structural differences likely affect how Mre11 from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with the exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on single-stranded and double-stranded DNA substrates, respectively.
View details for DOI 10.1016/j.jmb.2010.01.049
View details for Web of Science ID 000276177300003
View details for PubMedID 20122942
View details for PubMedCentralID PMC2839085
Pleckstrin homology (PH) domains have been identified only in eukaryotic proteins to date. We have determined crystal structures for three members of an uncharacterized protein family (Pfam PF08000), which provide compelling evidence for the existence of PH-like domains in bacteria (PHb). The first two structures contain a single PHb domain that forms a dome-shaped, oligomeric ring with C(5) symmetry. The third structure has an additional helical hairpin attached at the C-terminus and forms a similar but much larger ring with C(12) symmetry. Thus, both molecular assemblies exhibit rare, higher-order, cyclic symmetry but preserve a similar arrangement of their PHb domains, which gives rise to a conserved hydrophilic surface at the intersection of the beta-strands of adjacent protomers that likely mediates protein-protein interactions. As a result of these structures, additional families of PHb domains were identified, suggesting that PH domains are much more widespread than originally anticipated. Thus, rather than being a eukaryotic innovation, the PH domain superfamily appears to have existed before prokaryotes and eukaryotes diverged.
View details for DOI 10.1016/j.jmb.2009.11.006
View details for Web of Science ID 000274766500004
View details for PubMedID 19913036
View details for PubMedCentralID PMC2817789
SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.
View details for DOI 10.1074/jbc.M109.018564
View details for Web of Science ID 000269734000060
View details for PubMedID 19567872
View details for PubMedCentralID PMC2757229
Cell-cycle-regulated stalk biogenesis in Caulobacter crescentus is controlled by a multistep phosphorelay system consisting of the hybrid histidine kinase ShkA, the histidine phosphotransfer (HPt) protein ShpA, and the response regulator TacA. ShpA shuttles phosphoryl groups between ShkA and TacA. When phosphorylated, TacA triggers a downstream transcription cascade for stalk synthesis in an RpoN-dependent manner. The crystal structure of ShpA was determined to 1.52 A resolution. ShpA belongs to a family of monomeric HPt proteins that feature a highly conserved four-helix bundle. The phosphorylatable histidine His56 is located on the surface of the helix bundle and is fully solvent exposed. One end of the four-helix bundle in ShpA is shorter compared with other characterized HPt proteins, whereas the face that potentially interacts with the response regulators is structurally conserved. Similarities of the interaction surface around the phosphorylation site suggest that ShpA is likely to share a common mechanism for molecular recognition and phosphotransfer with yeast phosphotransfer protein YPD1 despite their low overall sequence similarity.
View details for DOI 10.1016/j.jmb.2009.05.023
View details for Web of Science ID 000268519200009
View details for PubMedID 19450606
View details for PubMedCentralID PMC2726009
ECX21941 represents a very large family (over 600 members) of novel, ocean metagenome-specific proteins identified by clustering of the dataset from the Global Ocean Sampling expedition. The crystal structure of ECX21941 reveals unexpected similarity to Sm/LSm proteins, which are important RNA-binding proteins, despite no detectable sequence similarity. The ECX21941 protein assembles as a homopentamer in solution and in the crystal structure when expressed in Escherichia coli and represents the first pentameric structure for this Sm/LSm family of proteins, although the actual oligomeric form in vivo is currently not known. The genomic neighborhood analysis of ECX21941 and its homologs combined with sequence similarity searches suggest a cyanophage origin for this protein. The specific functions of members of this family are unknown, but our structure analysis of ECX21941 indicates nucleic acid-binding capabilities and suggests a role in RNA and/or DNA processing.
View details for DOI 10.1002/prot.22360
View details for Web of Science ID 000264169400004
View details for PubMedID 19173316
View details for PubMedCentralID PMC2785455
The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 A resolution, respectively, and contain a bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as gamma-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.
View details for DOI 10.1016/j.str.2008.12.008
View details for PubMedID 19217401
Regulatory inactivation of DnaA is dependent on Hda (homologous to DnaA), a protein homologous to the AAA+ (ATPases associated with diverse cellular activities) ATPase region of the replication initiator DnaA. When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA-ATP into inactive DnaA-ADP. The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 A resolution reveals that Hda resembles typical AAA+ ATPases. The arrangement of the two subdomains in Hda (residues 1-174 and 175-241) differs dramatically from that of DnaA. A CDP molecule anchors the Hda domains in a conformation that promotes dimer formation. The Hda dimer adopts a novel oligomeric assembly for AAA+ proteins in which the arginine finger, crucial for ATP hydrolysis, is fully exposed and available to hydrolyze DnaA-ATP through a typical AAA+ type of mechanism. The sliding clamp binding motifs at the N-terminus of each Hda monomer are partially buried and combine to form an antiparallel beta-sheet at the dimer interface. The inaccessibility of the clamp binding motifs in the CDP-bound structure of Hda suggests that conformational changes are required for Hda to form a functional complex with the clamp. Thus, the CDP-bound Hda dimer likely represents an inactive form of Hda.
View details for DOI 10.1016/j.jmb.2008.10.059
View details for Web of Science ID 000262916900004
View details for PubMedID 19000695
View details for PubMedCentralID PMC2667141
TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.
View details for DOI 10.1002/prot.21673
View details for PubMedID 17654547
BtDyP from Bacteroides thetaiotaomicron (strain VPI-5482) and TyrA from Shewanella oneidensis are dye-decolorizing peroxidases (DyPs), members of a new family of heme-dependent peroxidases recently identified in fungi and bacteria. Here, we report the crystal structures of BtDyP and TyrA at 1.6 and 2.7 A, respectively. BtDyP assembles into a hexamer, while TyrA assembles into a dimer; the dimerization interface is conserved between the two proteins. Each monomer exhibits a two-domain, alpha+beta ferredoxin-like fold. A site for heme binding was identified computationally, and modeling of a heme into the proposed active site allowed for identification of residues likely to be functionally important. Structural and sequence comparisons with other DyPs demonstrate a conservation of putative heme-binding residues, including an absolutely conserved histidine. Isothermal titration calorimetry experiments confirm heme binding, but with a stoichiometry of 0.3:1 (heme:protein).
View details for DOI 10.1002/prot.21550
View details for PubMedID 17654545