Bio

Honors & Awards


  • Graduate Research Fellowship, National Science Foundation (9/1/2010 - 9/1/2013)

Professional Education


  • Doctor of Philosophy, Stanford University, SBIO-PHD (2014)
  • Bachelor of Mathematics, University of Minnesota Twin Cities, Mathematics (2009)
  • Bachelor of Science, University of Minnesota Twin Cities, Biochemistry (2009)

Research & Scholarship

Lab Affiliations


Publications

Journal Articles


  • Activation and allosteric modulation of a muscarinic acetylcholine receptor NATURE Kruse, A. C., Ring, A. M., Manglik, A., Hu, J., Hu, K., Eitel, K., Huebner, H., Pardon, E., Valant, C., Sexton, P. M., Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J., Weis, W. I., Garcia, K. C., Wess, J., Kobilka, B. K. 2013; 504 (7478): 101-?

    Abstract

    Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.

    View details for DOI 10.1038/nature12735

    View details for Web of Science ID 000327851700039

    View details for PubMedID 24256733

  • Novel Tripod Amphiphiles for Membrane Protein Analysis CHEMISTRY-A EUROPEAN JOURNAL Chae, P. S., Kruse, A. C., Gotfryd, K., Rana, R. R., Cho, K. H., Rasmussen, S. G., Bae, H. E., Chandra, R., Gether, U., Guan, L., Kobilka, B. K., Loland, C. J., Byrne, B., Gellman, S. H. 2013; 19 (46): 15645-15651
  • Applications of molecular replacement to G protein-coupled receptors ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Kruse, A. C., Manglik, A., Kobilka, B. K., Weis, W. I. 2013; 69: 2287-2292

    Abstract

    G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

    View details for DOI 10.1107/S090744491301322X

    View details for Web of Science ID 000326648900016

    View details for PubMedID 24189241

  • Adrenaline-activated structure of ß2-adrenoceptor stabilized by an engineered nanobody. Nature Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., Garcia, K. C., Kobilka, B. K. 2013; 502 (7472): 575-579

    Abstract

    G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

    View details for DOI 10.1038/nature12572

    View details for PubMedID 24056936

  • Muscarinic Receptors as Model Targets and Antitargets for Structure-Based Ligand Discovery MOLECULAR PHARMACOLOGY Kruse, A. C., Weiss, D. R., Rossi, M., Hu, J., Hu, K., Eitel, K., Gmeiner, P., Wess, J., Kobilka, B. K., Shoichet, B. K. 2013; 84 (4): 528-540
  • Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature Shukla, A. K., Manglik, A., Kruse, A. C., Xiao, K., Reis, R. I., Tseng, W., Staus, D. P., Hilger, D., Uysal, S., Huang, L., Paduch, M., Tripathi-Shukla, P., Koide, A., Koide, S., Weis, W. I., Kossiakoff, A. A., Kobilka, B. K., Lefkowitz, R. J. 2013; 497 (7447): 137-141

    Abstract

    The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ?-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ?-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ?-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ?-arrestin-1. The structure of the ?-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ?-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ?-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ?-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

    View details for DOI 10.1038/nature12120

    View details for PubMedID 23604254

  • Glucose-Neopentyl Glycol (GNG) amphiphiles for membrane protein study CHEMICAL COMMUNICATIONS Chae, P. S., Rana, R. R., Gotfryd, K., Rasmussen, S. G., Kruse, A. C., Cho, K. H., Capaldi, S., Carlsson, E., Kobilka, B., Loland, C. J., Gether, U., Banerjee, S., Byrne, B., Lee, J. K., Gellman, S. H. 2013; 49 (23): 2287-2289

    Abstract

    The development of a new class of surfactants for membrane protein manipulation, "GNG amphiphiles", is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. (Science, 2012, 337, 473).

    View details for DOI 10.1039/c2cc36844g

    View details for Web of Science ID 000315169400003

    View details for PubMedID 23165475

  • Muscarinic Receptors as Model Targets and Antitargets forStructure-Based Ligand Discovery. Molecular pharmacology Kruse, A. C., Weiss, D. R., Rossi, M., Hu, J., Hu, K., Eitel, K., Gmeiner, P., Wess, J., Kobilka, B. K., Shoichet, B. K. 2013

    Abstract

    G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacological profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and almost all are cationic in nature. Using structure-based docking against the M2 and M3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype-selectivities. Of 19 docking-prioritized molecules tested against the M2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar Ki values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M2 and M3 receptors, we selected molecules predicted by docking to bind to the M3 and but not the M2 receptor. Of 16 molecules tested, eight bound to the M3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M3 receptor without measurable M2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse b-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biological functions, even in an area as heavily explored as muscarinic pharmacology.

    View details for PubMedID 23887926

  • A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins CHEMISTRY-A EUROPEAN JOURNAL Chae, P. S., Rasmussen, S. G., Rana, R. R., Gotfryd, K., Kruse, A. C., Manglik, A., Cho, K. H., Nurva, S., Gether, U., Guan, L., Loland, C. J., Byrne, B., Kobilka, B. K., Gellman, S. H. 2012; 18 (31): 9485-9490

    View details for DOI 10.1002/chem.201200069

    View details for Web of Science ID 000306670200005

    View details for PubMedID 22730191

  • Crystal structure of the mu-opioid receptor bound to a morphinan antagonist NATURE Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Mathiesen, J. M., Sunahara, R. K., Pardo, L., Weis, W. I., Kobilka, B. K., Granier, S. 2012; 485 (7398): 321-U170

    Abstract

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8?Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

    View details for DOI 10.1038/nature10954

    View details for Web of Science ID 000304099100032

    View details for PubMedID 22437502

  • Structure of the delta-opioid receptor bound to naltrindole NATURE Granier, S., Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Weis, W. I., Kobilka, B. K. 2012; 485 (7398): 400-U171

    Abstract

    The opioid receptor family comprises three members, the µ-, ?- and ?-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The ?-opioid receptor (?-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the µ-OR and ?-OR have recently been solved. Here we report the crystal structure of the mouse ?-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the µ-OR and ?-OR, the ?-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the ?-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

    View details for DOI 10.1038/nature11111

    View details for Web of Science ID 000304099100049

    View details for PubMedID 22596164

  • Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist NATURE Haga, K., Kruse, A. C., Asada, H., Yurugi-Kobayashi, T., Shiroishi, M., Zhang, C., Weis, W. I., Okada, T., Kobilka, B. K., Haga, T., Kobayashi, T. 2012; 482 (7386): 547-U147

    Abstract

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

    View details for DOI 10.1038/nature10753

    View details for Web of Science ID 000300770500055

    View details for PubMedID 22278061

  • Structure and dynamics of the M3 muscarinic acetylcholine receptor NATURE Kruse, A. C., Hu, J., Pan, A. C., Arlow, D. H., Rosenbaum, D. M., Rosemond, E., Green, H. F., Liu, T., Chae, P. S., Dror, R. O., Shaw, D. E., Weis, W. I., Wess, J., Kobilka, B. K. 2012; 482 (7386): 552-556

    Abstract

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

    View details for DOI 10.1038/nature10867

    View details for Web of Science ID 000300770500056

    View details for PubMedID 22358844

  • T Cell Receptor Signaling Is Limited by Docking Geometry to Peptide-Major Histocompatibility Complex IMMUNITY Adams, J. J., Narayanan, S., Liu, B., Birnbaum, M. E., Kruse, A. C., Bowerman, N. A., Chen, W., Levin, A. M., Connolly, J. M., Zhu, C., Kranz, D. M., Garcia, K. C. 2011; 35 (5): 681-693

    Abstract

    T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

    View details for DOI 10.1016/j.immuni.2011.09.013

    View details for Web of Science ID 000297390800009

    View details for PubMedID 22101157

  • 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 Å 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

  • Structural Basis of Specificity and Cross-Reactivity in T Cell Receptors Specific for Cytochrome c-I-E-k JOURNAL OF IMMUNOLOGY Newell, E. W., Ely, L. K., Kruse, A. C., Reay, P. A., Rodriguez, S. N., Lin, A. E., Kuhns, M. S., Garcia, K. C., Davis, M. M. 2011; 186 (10): 5823-5832

    Abstract

    T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions.

    View details for DOI 10.4049/jimmunol.1100197

    View details for Web of Science ID 000290150700034

    View details for PubMedID 21490152

  • Structure of a mutant beta toxin from Staphylococcus aureus reveals domain swapping and conformational flexibility ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS Kruse, A. C., Huseby, M. J., Shi, K., Digre, J., Ohlendorf, D. H., Earhart, C. A. 2011; 67: 438-441

    Abstract

    The 3.35?Å resolution crystal structure of a mutant form of the staphylococcal sphingomyelinase ? toxin in which a conserved hydrophobic ?-hairpin has been deleted is reported. It is shown that this mutation induces domain swapping of a C-terminal ?-strand, leading to the formation of dimers linked by a conformationally flexible hinge region. Eight dimers are seen in the asymmetric unit, exhibiting a broad spectrum of conformations trapped in place by intermolecular contacts within the crystal lattice. Furthermore, the 16 monomers within each asymmetric unit exhibit a remarkable heterogeneity in thermal factors, which can be accounted for by the varying degrees to which each monomer interacts with other molecules in the crystal. This structure provides a unique example of the challenges associated with crystallographic study of flexible proteins.

    View details for DOI 10.1107/S1744309111005239

    View details for Web of Science ID 000289738400004

    View details for PubMedID 21505235

  • Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins NATURE METHODS Chae, P. S., Rasmussen, S. G., Rana, R. R., Gotfryd, K., Chandra, R., Goren, M. A., Kruse, A. C., Nurva, S., Loland, C. J., Pierre, Y., Drew, D., Popot, J., Picot, D., Fox, B. G., Guan, L., Gether, U., Byrne, B., Kobilka, B., Gellman, S. H. 2010; 7 (12): 1003-U90

    Abstract

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

    View details for DOI 10.1038/NMETH.1526

    View details for Web of Science ID 000284686300016

    View details for PubMedID 21037590

  • Negative Selection and Peptide Chemistry Determine the Size of Naive Foreign Peptide-MHC Class II-Specific CD4(+) T Cell Populations JOURNAL OF IMMUNOLOGY Chu, H. H., Moon, J. J., Kruse, A. C., Pepper, M., Jenkins, M. K. 2010; 185 (8): 4705-4713

    Abstract

    Naive CD4(+) T cell populations that express TCRs specific for different foreign peptide-MHC class II complex (pMHCII) ligands can vary in size over several orders of magnitude. This variation may explain why immune responses to some peptides are stronger than others. In this study, we used a sensitive pMHCII-tetramer-based cell enrichment method to study the derivation of two naive foreign pMHCII-specific naive CD4(+) T cell populations that differed in size by 8-fold in normal mice. Analysis of mice in which thymic negative selection was impaired revealed that the smaller population underwent more clonal deletion than the larger population. In addition, large naive cell populations tended to recognize peptides with tryptophan residues as TCR contacts. Thus, the foreign pMHCII that tend to be recognized by large naive populations induce minimal clonal deletion and contain certain amino acids with the capacity to interact favorably with TCRs.

    View details for DOI 10.4049/jimmunol.1002276

    View details for Web of Science ID 000282525900023

    View details for PubMedID 20861357

  • Beta toxin catalyzes formation of nucleoprotein matrix in staphylococcal biofilms PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huseby, M. J., Kruse, A. C., Digre, J., Kohler, P. L., Vocke, J. A., Mann, E. E., Bayles, K. W., Bohach, G. A., Schlievert, P. M., Ohlendorf, D. H., Earhart, C. A. 2010; 107 (32): 14407-14412

    Abstract

    Biofilms are surface-associated communities of microbes encompassed by an extracellular matrix. It is estimated that 80% of all bacterial infections involve biofilm formation, but the structure and regulation of biofilms are incompletely understood. Extracellular DNA (eDNA) is a major structural component in many biofilms of the pathogenic bacterium Staphylococcus aureus, but its role is enigmatic. Here, we demonstrate that beta toxin, a neutral sphingomyelinase and a virulence factor of S. aureus, forms covalent cross-links to itself in the presence of DNA (we refer to this as biofilm ligase activity, independent of sphingomyelinase activity) producing an insoluble nucleoprotein matrix in vitro. Furthermore, we show that beta toxin strongly stimulates biofilm formation in vivo as demonstrated by a role in causation of infectious endocarditis in a rabbit model. Together, these results suggest that beta toxin cross-linking in the presence of eDNA assists in forming the skeletal framework upon which staphylococcal biofilms are established.

    View details for DOI 10.1073/pnas.0911032107

    View details for Web of Science ID 000280767700078

    View details for PubMedID 20660751

  • X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal PROTEIN SCIENCE Hellberg, K., Grimsrud, P. A., Kruse, A. C., Banaszak, L. J., Ohlendorf, D. H., Bernlohr, D. A. 2010; 19 (8): 1480-1489

    Abstract

    Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long-chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4-hydroxy-2-nonenal (4-HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4-HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4-HNE have been solved to 1.9 A and 2.3 A resolution, respectively. While the 4-HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4-HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4-HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.

    View details for DOI 10.1002/pro.427

    View details for Web of Science ID 000280481300004

    View details for PubMedID 20509169

  • Identification and Characterization of a Small Molecule Inhibitor of Fatty Acid Binding Proteins JOURNAL OF MEDICINAL CHEMISTRY Hertzel, A. V., Hellberg, K., Reynolds, J. M., Kruse, A. C., Juhlmann, B. E., Smith, A. J., Sanders, M. A., Ohlendorf, D. H., Suttles, J., Bernlohr, D. A. 2009; 52 (19): 6024-6031

    Abstract

    Molecular disruption of the lipid carrier AFABP/aP2 in mice results in improved insulin sensitivity and protection from atherosclerosis. Because small molecule inhibitors may be efficacious in defining the mechanism(s) of AFABP/aP2 action, a chemical library was screened and identified 1 (HTS01037) as a pharmacologic ligand capable of displacing the fluorophore 1-anilinonaphthalene 8-sulfonic acid from the lipid binding cavity. The X-ray crystal structure of 1 bound to AFABP/aP2 revealed that the ligand binds at a structurally similar position to a long-chain fatty acid. Similar to AFABP/aP2 knockout mice, 1 inhibits lipolysis in 3T3-L1 adipocytes and reduces LPS-stimulated inflammation in cultured macrophages. 1 acts as an antagonist of the protein-protein interaction between AFABP/aP2 and hormone sensitive lipase but does not activate PPARgamma in macrophage or CV-1 cells. These results identify 1 as an inhibitor of fatty acid binding and a competitive antagonist of protein-protein interactions mediated by AFABP/aP2.

    View details for DOI 10.1021/jm900720m

    View details for Web of Science ID 000270361600024

    View details for PubMedID 19754198

  • Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab(-/-) mice PEPTIDES Yoder, A. R., Kruse, A. C., Earhart, C. A., Ohlendorf, D. H., Potter, L. R. 2008; 29 (9): 1575-1581

    Abstract

    C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.

    View details for DOI 10.1016/j.peptides.2008.04.020

    View details for Web of Science ID 000259346400015

    View details for PubMedID 18554750

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