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Research Interests: Immunology, Biology, Medicine, Protein Structure and Function, Humans, and 15 moreSequence alignment, Mice, Adaptive Immunity, Animals, Cytomegalovirus, Immune system, Rats, Major histocompatibility complex, Host Pathogen Interactions, Amino Acid Sequence, NK Cell, MHC class I, Host Defense, Molecular Sequence Data, and Immunologic
A valuable model for studying virus-host interactions is mouse cytomegalovirus (MCMV), which encodes the m145 family of glycoproteins with predicted MHC-I structure. m153, expressed independently of beta-2 microglobulin and peptide, and... more
A valuable model for studying virus-host interactions is mouse cytomegalovirus (MCMV), which encodes the m145 family of glycoproteins with predicted MHC-I structure. m153, expressed independently of beta-2 microglobulin and peptide, and is found on the surface of infected cells by flow cytometry. We constructed a reporter cell to identify tissues expressing the ligand of m153 by preparing the DNA encoding the extracellular domains of m153 linked to human zeta chain, and introducing this construct into 43.1 cells that fluoresce green on activation. We identified CD11c+ splenic dendritic cells (DC) and bone marrow DC as potent stimulators. Further characterization of these cells via multi-color staining with an m153 tetramer, and DC markers and antibody blocking experiments suggest a possible candidate for the m153 ligand. This may contribute to our understanding of the steps in virus lifecycle, and shed light on the evolution of viral immunoevasins.
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MHC class I (MHC-I) molecules bound to antigenic peptides form the molecular structures recognized by MHC-I-restricted CD8+ T lymphocytes. On binding an antigenic peptide, the newly synthesized MHC-I sequesters a previously exposed region... more
MHC class I (MHC-I) molecules bound to antigenic peptides form the molecular structures recognized by MHC-I-restricted CD8+ T lymphocytes. On binding an antigenic peptide, the newly synthesized MHC-I sequesters a previously exposed region of the alpha-1 domain including residues 46-52, is released from tapasin, and proceeds to the cell surface. However, the changes in shape of MHC-I on peptide binding and release from tapasin remain unclear. We have determined four X-ray structures (from 1.64Å to 1.87 Å resolution) of an overlapping family of peptides from the H-2Ld alpha-1 domain (including residues 46-52) bound to the Fab fragment of 64-3-7, a mAb that reacts with peptide-receptive (PR) but not with peptide-loaded (PL) H-2Ld MHC-I. Positional differences of backbone and sidechain atoms of this alpha-1 domain segment, compared to the same segment in PL H-2Ld, indicate a significant conformational change on peptide loading. Among other changes, the side chain of the invariant Trp51 residue, and of Met52, although buried in PL MHCI, form extensive noncovalent bonds with mAb 64-3-7, indicating an external (solvent exposed) location in PR MHC-I. Furthermore, the 3(10) helix present in this segment of PL MHC-I is also present in the free peptide bound to MAb 64-3-7, and hence in the PR molecule. These findings help to delineate and provide insight into the conformational changes in MHC-I that accompany peptide loading. Supported by the intramural research program of the NIAID.
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Molecules encoded by the Major Histocompatibility Complex (MHC) bind self or foreign peptides and display these at the cell surface for recognition by receptors on T lymphocytes (designated T cell receptors-TCR) or on natural killer (NK)... more
Molecules encoded by the Major Histocompatibility Complex (MHC) bind self or foreign peptides and display these at the cell surface for recognition by receptors on T lymphocytes (designated T cell receptors-TCR) or on natural killer (NK) cells. These ligand/receptor interactions govern T cell and NK cell development as well as activation of T memory and effector cells. Such cells participate in immunological processes that regulate immunity to various pathogens, resistance and susceptibility to cancer, and autoimmunity. The past few decades have witnessed the accumulation of a huge knowledge base of the molecular structures of MHC molecules bound to numerous peptides, of TCRs with specificity for many different peptide/MHC (pMHC) complexes, of NK cell receptors (NKR), of MHC-like viral immunoevasins, and of pMHC/TCR and pMHC/NKR complexes. This chapter reviews the structural principles that govern peptide/MHC (pMHC), pMHC/TCR, and pMHC/NKR interactions, for both MHC class I (MHC-I) and MHC class II (MHC-II) molecules. In addition, we discuss the structures of several representative MHC-like molecules. These include host molecules that have distinct biological functions, as well as virus-encoded molecules that contribute to the evasion of the immune response.
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To stimulate T cells, antigenic peptides must not only bind the presenting MHC molecule, but must form a complex of sufficient stability to interact with the T cell receptor (TCR). In a murine autoimmune gastritis model based on the... more
To stimulate T cells, antigenic peptides must not only bind the presenting MHC molecule, but must form a complex of sufficient stability to interact with the T cell receptor (TCR). In a murine autoimmune gastritis model based on the transgenic expression of MHC-II restricted T cell receptors (TCR) directed against peptides from the alpha chain of the gastric parietal cell proton pump, the H/K ATPase, previous studies mapped residues 630-641 (PITAKAIAASVG), and residues 889-900 (PLLCVGLRPQWE) as the epitopes recognized by TXA23 and TXA51 transgenic T cells respectively. We studied the dissociation kinetics of peptides with various amino acid substitutions (including alanine-substitution at each non-alanine position). Binding to IAd was compared to T cell stimulatory activity of the same peptides in MHC-peptide complexes. Dissociation half lives at pH 5.3 (similar to endosomes) varied from ~20 hours to over 600 hrs. Peptide/MHC stability is greatest at neutral pH, where a nonstimulatory peptide (CLIP) and a highly stimulatory peptide (GHNLAVGLRPQWHE) had half lives approximately tenfold longer than at pH5.3. We find that a half-life of >20 hours at pH 5.3 is sufficient for a peptide with the necessary epitopic residues to retain immunogenicity. These results can be explained in the context of the X-ray structure of the IAd/PLLCVGLRPQWE complex. Supported by the NIAID intramural research program.
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We have developed a murine model of autoimmune gastritis by generating two TCR transgenic lines expressing I-Ad restricted TCRs specific for two different peptide epitopes of gastric H/K ATPase. Both lines spontaneously develop AIG but... more
We have developed a murine model of autoimmune gastritis by generating two TCR transgenic lines expressing I-Ad restricted TCRs specific for two different peptide epitopes of gastric H/K ATPase. Both lines spontaneously develop AIG but with differing incidence, severity, and kinetics. The Tx23 line develops disease early, severely, with 100% incidence and a predominantly Th1 phenotype. The Tx51 line develops disease only after two months, only 50–80% of the mice are affected. and is predominantly of the Th2 type. To investigate the role of TCR affinity in the disease phenotypes in these mice, we generated recombinant, soluble forms of the Tx23 and Tx51 TCRs, and monomeric and multimeric forms of their cognate I-Ad/peptide complexes. Peptide dose response experiments suggest that the Tx51 TCR is of higher functional avidity than the Tx23 TCR, suggesting that decreased peptide availability in vivo may limit the activation of Tx51 T cells. Surface plasmon resonance (SPR) studies revealed specific and dose dependent binding when multimeric but not monomeric I-Ad/peptide complexes were offered to the TCR, consistent with the relatively weak affinities of these autoreactive TCRs. These experiments exploit strategies that may be employed to detect binding of low affinity TCRs to their MHC/peptide ligands. This was supported by the Intramural Research Program of the NIAID.
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Mouse cytomegalovirus (MCMV) is a valuable model for studying virus-host interactions and mechanisms of viral immune evasion. MCMV encodes the m145 family of glycoproteins with predicted MHC-I structure. Among these, m153 is expressed on... more
Mouse cytomegalovirus (MCMV) is a valuable model for studying virus-host interactions and mechanisms of viral immune evasion. MCMV encodes the m145 family of glycoproteins with predicted MHC-I structure. Among these, m153 is expressed on the surface of infected cells predominantly during the early phases of infection, independent of β2 microglobulin and peptide. We previously determined the X-ray structure of m153, establishing that it has an MHC-I-like fold and forms a stable non-covalent dimer. To identify immune cells that might express a ligand for m153, we constructed a reporter cell in which the extracellular domains of m153 were linked to human ζ chain driving green fluorescent protein upon activation. Using the reporter cell assay, we observed that CD11c+ splenic dendritic cells (DCs) and bone marrow DCs potently stimulate the m153 reporter cell. Further characterization of these cells by flow cytometry staining with an m153 tetramer and a battery of DC markers, as well as antibody blocking experiments, suggested several candidates for the m153 ligand. Transfectant cells and gene knockout animals are being studied to confirm the identity of these candidates. The identification of a host cell ligand for m153 should improve our understanding of mechanisms of host immune suppression during viral infection and will provide insight on the evolution of viral immune evasion.
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The mouse cytomegalovirus (mCMV), a β-herpesvirus that encodes more than 170 open reading frames (ORFs), serves as a model for infection by the human CMV. Protein homology suggests that at least 10 of the mCMV ORFs might be related in... more
The mouse cytomegalovirus (mCMV), a β-herpesvirus that encodes more than 170 open reading frames (ORFs), serves as a model for infection by the human CMV. Protein homology suggests that at least 10 of the mCMV ORFs might be related in structure to MHC-I molecules. Several of these putative viral MHC-I like molecules (MHC-Iv) have been shown to function as immunoevasins. To establish a structural basis for the function and evolution of MHC-Iv molecules, we have initiated a structural survey, and report here the X-ray crystallographic structures of two mCMV molecules, m144 and m153. m144 crystallized in complex with the light chain, β2-microglobulin (β-2m), and its structure was determined to 1.9 Å resolution by molecular replacement. m153 formed fragile crystals that diffracted to 2.4 Å and its structure was solved by the single anomalous dispersion method. Both molecules reveal readily identifiable α1 and α2 helices and Ig-like α3 domains. Neither molecule has any bound peptide or other small molecule ligand. m144 interacts with β2-m, while m153 lacks a light chain. m153, which purifies as a non-covalently associated dimer, reveals an extended amino terminus stabilized by a unique disulfide bond, and has a lengthy α2 helix that extends into the α3 domain. The m153 dimer is confirmed by sedimentation analysis. Comparison of m144 and m153 structures reveals conservation in the α3 domain, and shows unique features in both α1 and α2. These structures offer insight into the evolution of MHC-Iv molecules and illustrate the versatility of the MHC-I fold. This research was supported in part by the Intramural Research Program of the NIH, NIAID.
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We have previously reported the development of mouse models of autoimmune gastritis in which BALB/c mice expressing transgenic αβ TCR, designated A23 and A51, spontaneously develop acute Th1-mediated high-penetrance and chronic... more
We have previously reported the development of mouse models of autoimmune gastritis in which BALB/c mice expressing transgenic αβ TCR, designated A23 and A51, spontaneously develop acute Th1-mediated high-penetrance and chronic Th2-mediated low penetrance forms of the disease, respectively. Using an array of synthetic mutated peptides in functional and MHC II-binding assays, we now report the core peptides recognized by each of these TCR. A23 recognizes H/K ATPase residues 633-641 (AKAIAASVG) and A51 sees 892-900 (CVGLRPQWE), both exhibiting a canonical 1, 4, 6, 9 I-Ad -binding motif. To extend these findings, we determined the X-ray crystallographic structure of complexes of I-Ad with both covalent and non-covalent peptides that stimulate the A51 TCR, and used these structures to generate a molecular model of the A23-stimulating peptide/I-Ad complex. The structural identification of peptide anchor residues in individual I-Ad binding pockets and of solvent exposed side chains as T cell epitopic residues readily explains T cell proliferation data and I-Ad/peptide off-rate measurements of the large panel of truncated, chimeric, and alanine or glycine-substituted synthetic peptides. The first structure of I-Ad in complex with an autoimmune peptide offers new insight in the general question of the structural basis of responses to self antigens.
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The cell surface expression of MHC-I molecules displaying peptides derived from intracellular proteolytic processing is a critical requirement for T cell-mediated immunity. A complex series of steps, guided by the peptide loading... more
The cell surface expression of MHC-I molecules displaying peptides derived from intracellular proteolytic processing is a critical requirement for T cell-mediated immunity. A complex series of steps, guided by the peptide loading machinery in the endoplasmic reticulum, ensures that only stably folded MHC-I molecules carrying peptides of proper length and high affinity are transported for display at the cell surface. TAP-binding protein, related (TAPBPR), has been recently shown to directly bind to certain MHC-I alleles and to facilitate peptide loading. To elucidate mechanistic details, we have examined the binding interaction between purified recombinant TAPBPR and MHC-I molecules loaded with truncated peptides that leave a portion of the peptide binding groove unoccupied. Such MHC-I molecules interact with TAPBPR with nanomolar affinities as measured by surface plasmon resonance and these TAPBPR/MHC-I complexes dissociate when offered a high affinity peptide. An extensive crystall...
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TAPBPR (TAP Binding Protein-Related), a tapasin homolog, functions as a peptide editor independent of the classical peptide loading complex (PLC) that consists of tapasin, transporter associated with antigen processing (TAP), ERp57, and... more
TAPBPR (TAP Binding Protein-Related), a tapasin homolog, functions as a peptide editor independent of the classical peptide loading complex (PLC) that consists of tapasin, transporter associated with antigen processing (TAP), ERp57, and calreticulin. Using insect-cell expressed recombinant luminal human TAPBPR, we previously reported interaction with HLA-A*02:01 molecules freed of peptide following photolysis of HLA-A2/beta2m complexes prepared with photolabile peptides. Here, we extend our analysis of the TABPBPR/MHC interaction by examining the binding of a set of MHC/peptide complexes prepared with different MHC-I molecules refolded with a variety of peptides. Using surface plasmon resonance, we show that a variety of refolded complexes, with or without photolysis of the bound peptide, bind to TAPBPR, raising the possibility that TAPBPR may interact either with a small proportion of peptide free molecules found in some MHC/peptide preparations, or with molecular conformations con...
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Adverse drug reactions (ADR) are a major obstacle to drug development. Genome-wide association studies identified several human leukocyte antigens (HLA)-class I alleles as risk factors for ADR. The HLA-B*57:01 allele has been found to be... more
Adverse drug reactions (ADR) are a major obstacle to drug development. Genome-wide association studies identified several human leukocyte antigens (HLA)-class I alleles as risk factors for ADR. The HLA-B*57:01 allele has been found to be associated with the development of abacavir (ABC) hypersensitivity syndrome (AHS) and with the induction of liver injury by the β-lactam antibiotic flucloxacillin. The nucleoside analog ABC, an inhibitor of the HIV reverse transcriptase, can induce severe multi-organ toxicity in >50% of HLA-B*57:01+ patients with HIV infection. In a previous study we showed that ABC induced binding to the HLA-B*57:01 of altered self-peptides containing predominantly isoleucine or leucine residues at the carboxyl terminus. Recognition of these self-peptides drives in vitro activation of cytotoxic CD8+ T cells. However, the early immune events/danger signals required to overcome the immune tolerance that otherwise suppress ADR are still unknown. In order to study H...
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The loading of MHC-I molecules with peptides for cell surface display is a crucial step in self-tolerance and activation of CD8 T cells. We studied TAPBPR (TAP binding protein-related) protein, a tapasin homolog, which is widely expressed... more
The loading of MHC-I molecules with peptides for cell surface display is a crucial step in self-tolerance and activation of CD8 T cells. We studied TAPBPR (TAP binding protein-related) protein, a tapasin homolog, which is widely expressed and IFN-γ inducible, but is not part of the classical MHC-I peptide-loading complex. We produced recombinant soluble TAPBPR and evaluated its interactions with several recombinant MHC-I molecules in vitro, by gel-shift, size exclusion chromatography, ultracentrifugation, and surface plasmon resonance. We show that TAPBPR binds MHC-I after photolysis of a bound peptide, and that the TAPBPR/MHC-I complex is dissociated by exposure to peptides that bind the MHC-I molecule, indicating a role of TAPBPR in stabilizing a peptide-receptive form of the MHC-I/β2m complex. Peptide-dependent release of MHC-I from TAPBPR is directly proportional to the peptide’s affinity for MHC-I. Peptide binding experiments indicate a role for TAPBPR in selection of high affi...
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The molecular mechanism by which MHC-I molecules are loaded with peptides is crucial to fundamental aspects of antigen presentation relating to self-tolerance and CD8 T cell activation. To gain insight into the mechanism of MHC-I peptide... more
The molecular mechanism by which MHC-I molecules are loaded with peptides is crucial to fundamental aspects of antigen presentation relating to self-tolerance and CD8 T cell activation. To gain insight into the mechanism of MHC-I peptide loading we have studied human TAPBPR (TAP binding protein-related) protein, which is 22% identical to tapasin in amino acid sequence, widely expressed and IFN-γ inducible, but is not part of the peptide-loading complex (PLC). To understand the interaction of TAPBPR with MHC-I, we produced recombinant soluble TAPBPR and evaluated its interactions with MHC-I/β2m complexes in vitro. Using recombinant MHC-I molecules refolded with photolabile peptides, we show, by gel-shift analysis, size exclusion chromatography, and analytical ultracentrifugation that TAPBPR binds MHC-I/β2m after photolysis of the bound peptide, in a 1:1 molar ratio. MHC-I molecules loaded with low-affinity peptides also bind TAPBPR. The TAPBPR/MHC interaction is reversed by exposure ...
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The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the... more
The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16–RBD and Sb45–RBD; a ternary complex of Sb45–RBD–Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design.One Sentence SummaryX-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details ...
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NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I–specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti–MHC-I mAb that blocks NK cell inhibitory... more
NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I–specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti–MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.
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Peptide loading of MHC-I molecules plays a critical role in the T cell response to infections and tumors as well as to interactions with inhibitory receptors on natural killer (NK) cells. To facilitate and optimize peptide acquisition,... more
Peptide loading of MHC-I molecules plays a critical role in the T cell response to infections and tumors as well as to interactions with inhibitory receptors on natural killer (NK) cells. To facilitate and optimize peptide acquisition, vertebrates have evolved specialized chaperones to stabilize MHC-I molecules during their biosynthesis and to catalyze peptide exchange favoring high affinity or optimal peptides to permit transport to the cell surface where stable peptide/MHC-I (pMHC-I) complexes are displayed and are available for interaction with T cell receptors and any of a host of inhibitory and activating receptors. Although components of the endoplasmic reticulum (ER) resident peptide loading complex (PLC) were identified some 30 years ago, the detailed biophysical parameters that govern peptide selection, binding, and surface display have recently been understood better with advances in structural methods including X-ray crystallography, cryogenic electron microscopy (cryo-EM...
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We have expressed in bacteria a single-chain T cell receptor (scTCR) with specificity for an HIV gp120-derived peptide bound to the murine MHC-I molecule, H-2Dd. This scTCR consists of V alpha covalently linked to the VbetaCbeta domains... more
We have expressed in bacteria a single-chain T cell receptor (scTCR) with specificity for an HIV gp120-derived peptide bound to the murine MHC-I molecule, H-2Dd. This scTCR consists of V alpha covalently linked to the VbetaCbeta domains that was solubilized, refolded, and purified in high yield. Specific binding of the scTCR to MHC/peptide complexes was demonstrated by surface plasmon resonance, with a Kd of 2 to 8 x 10(-6) M. This scTCR specifically inhibited T cell activation, and stained cell surface MHC/peptide complexes as measured by cytofluorimetry. The preservation of binding specificity by such a three-domain scTCR suggests that this structure is sufficient for specific MHC/peptide recognition and that this strategy will be of general use as applied to other TCR.
Research Interests: Immunology, Biology, Immunology of the Gut, Medicine, Circular Dichroism, and 15 moreMice, Animals, Monoclonal Antibodies, Peptides, Peptide, Protein Secondary Structure Prediction, Major histocompatibility complex, Receptor, Amino Acid Sequence, Base Sequence, Protein Binding, Epitopes, Immunoglobulins, Hybridomas, and Molecular Sequence Data
MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing αβ TCR. Although the activation of T cells is controlled by the interaction of the TCR with... more
MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing αβ TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 × 10−7 M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8α and β chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that som...
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Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed... more
Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin–MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies. The tapasin-stabilized peptide-receptive state of HLA-B*44:05 is characterized by distortion of the peptide binding groove and destabilization of the β2-microglobulin interaction, leading to release of peptide. Movements of the membrane proximal Ig-like domains of tapasin, HLA-B*44:05, and β2-microglobulin accompany the transition to a peptide-receptive state. Together this ensemble of crystal structures provides insights into a distinct mechanism of tapasin-mediated peptide exchange.
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Immune recognition by T lymphocytes and natural killer (NK) cells is in large part dependent on the identification of cell surface MHC molecules bearing peptides generated from either endogenous (MHC I) or exogenous (MHC II) dependent... more
Immune recognition by T lymphocytes and natural killer (NK) cells is in large part dependent on the identification of cell surface MHC molecules bearing peptides generated from either endogenous (MHC I) or exogenous (MHC II) dependent pathways. This review focuses on MHC I molecules that coordinately fold to bind self or foreign peptides for such surface display. Peptide loading occurs in an antigen presentation pathway that includes either the multimolecular peptide loading complex (PLC) or a single chain chaperone/catalyst, TAP binding protein, related, TAPBPR, that mimics a key component of the PLC, tapasin. Recent structural and dynamic studies of TAPBPR reveal details of its function and reflect on mechanisms common to tapasin. Regions of structural conservation among species suggest that TAPBPR and tapasin have evolved to satisfy functional complexities demanded by the enormous polymorphism of MHC I molecules. Recent studies suggest that these two chaperone/catalysts exploit s...
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Molecules encoded by the Major Histocompatibility Complex (MHC) bind self or foreign peptides and display these at the cell surface for recognition by receptors on T lymphocytes (designated T cell receptors-TCR) or on natural killer (NK)... more
Molecules encoded by the Major Histocompatibility Complex (MHC) bind self or foreign peptides and display these at the cell surface for recognition by receptors on T lymphocytes (designated T cell receptors-TCR) or on natural killer (NK) cells. These ligand/receptor interactions govern T cell and NK cell development as well as activation of T memory and effector cells. Such cells participate in immunological processes that regulate immunity to various pathogens, resistance and susceptibility to cancer, and autoimmunity. The past few decades have witnessed the accumulation of a huge knowledge base of the molecular structures of MHC molecules bound to numerous peptides, of TCRs with specificity for many different peptide/MHC (pMHC) complexes, of NK cell receptors (NKR), of MHC-like viral immunoevasins, and of pMHC/TCR and pMHC/NKR complexes. This chapter reviews the structural principles that govern peptide/MHC (pMHC), pMHC/TCR, and pMHC/NKR interactions, for both MHC class I (MHC-I) and MHC class II (MHC-II) molecules. In addition, we discuss the structures of several representative MHC-like molecules. These include host molecules that have distinct biological functions, as well as virus-encoded molecules that contribute to the evasion of the immune response.
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Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the... more
Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40–50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficien...
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CD4+ T cells that lead to autoimmune gastritis (AIG) in BALB/c mice are either Th1 or Th2 cells. To test whether the phenotype of disease is related to the particular TCR expressed by the pathogenic cell, we have generated several lines... more
CD4+ T cells that lead to autoimmune gastritis (AIG) in BALB/c mice are either Th1 or Th2 cells. To test whether the phenotype of disease is related to the particular TCR expressed by the pathogenic cell, we have generated several lines of TCR transgenic mice using receptors cloned from pathogenic Th1 or Th2 cells. We previously described spontaneous inflammatory AIG in A23 mice, caused by the transgenic expression of the TCR from a Th1 clone, TXA23. In this study we describe the generation of A51 mouse lines, transgenic for the TCR of a CD4+ self-reactive Th2 clone, TXA51. A proportion of A51 mice spontaneously develop AIG by 10 wk of age, with a disease characterized by eosinophilic infiltration of the gastric mucosa and Th2 differentiation of transgenic T cells in the gastric lymph node. The Th2 phenotype of this autoimmune response seems to be related to a low availability of MHC class II-self peptide complexes. This in vivo model of spontaneous Th2-mediated, organ-specific auto...
Research Interests: Cardiology, Immunology, Biology, Autoimmunity, Immunology of the Gut, and 15 moreMedicine, Logistic Regression, Cell Differentiation, Humans, Internal Medicine, Autoimmune diseases, Mice, Animals, Antigen Presentation, Amino Acid Sequence, Base Sequence, Gastritis, Severe combined immunodeficiency, Adoptive Transfer, and Molecular Sequence Data
The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established.... more
The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 ...
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Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus , is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major... more
Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus , is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and...
Research Interests: Biology, Cytokines, Medicine, Biological Sciences, Infection and immunity, and 15 moreHumans, Mice, Enterotoxins, Animals, Blood, Infection, Monoclonal Antibodies, Antibody, Major histocompatibility complex, Monoclonal Antibody, Antigen, Biochemistry and cell biology, immunoglobulin G, Enterotoxin, and Medical and Health Sciences
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Research Interests:
Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is... more
Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.
Research Interests:
Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression... more
Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression of NKG2D ligands. The mouse CMV MHC class I (MHC-I)–like m152/gp40 glycoprotein down-regulates retinoic acid early inducible-1 (RAE1) NKG2D ligands as well as host MHC-I. Here we describe the crystal structure of an m152/RAE1γ complex and confirm the intermolecular contacts by mutagenesis. m152 interacts in a pincer-like manner with two sites on the α1 and α2 helices of RAE1 reminiscent of the NKG2D interaction with RAE1. This structure of an MHC-I–like immunoevasin/MHC-I–like ligand complex explains the binding specificity of m152 for RAE1 and allows modeling of the interaction of m152 with classical MHC-I and of related viral immunoevasins.