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Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection.

ABSTRACT: Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.

SUBMITTER: Anjanappa R

PROVIDER: S-EPMC7066147 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Native mass spectrometry of peptide-free and partially loaded MHC class I molecules

Project description:A disulfide-stabilized MHC class I molecule (HLA-A*02:01(Y84C/A139C), short dsA2) was characterized by native mass spectrometry. dsA2 was shown to be structurally intact in absence of any peptide whereas its wild type form was not. Furthermore, it was observed that the mutation did not disrupt the binding of cognate peptides.

2022-05-11 | PXD016708 | Pride

The first step of peptide selection in antigen presentation by MHC class I molecules.

Project description:MHC class I molecules present a variable but limited repertoire of antigenic peptides for T-cell recognition. Understanding how peptide selection is achieved requires mechanistic insights into the interactions between the MHC I and candidate peptides. We find that, at first encounter, MHC I H-2K(b) considers a wide range of peptides, including those with expanded N termini and unfitting anchor residues. Discrimination occurs in the second step, when noncanonical peptides dissociate with faster exchange rates. This second step exhibits remarkable temperature sensitivity, as illustrated by numerous noncanonical peptides presented by H-2K(b) in cells cultured at 26 °C relative to 37 °C. Crystallographic analyses of H-2K(b)-peptide complexes suggest that a conformational adaptation of H-2K(b) drives the decisive step in peptide selection. We propose that MHC class I molecules consider initially a large peptide pool, subsequently refined by a temperature-sensitive induced-fit mechanism to retain the canonical peptide repertoire.

| S-EPMC4321303 | biostudies-literature

Whole genome duplications have provided teleosts with many roads to peptide loaded MHC class I molecules.

Project description:BackgroundIn sharks, chickens, rats, frogs, medaka and zebrafish there is haplotypic variation in MHC class I and closely linked genes involved in antigen processing, peptide translocation and peptide loading. At least in chicken, such MHCIa haplotypes of MHCIa, TAP2 and Tapasin are shown to influence the repertoire of pathogen epitopes being presented to CD8+ T-cells with subsequent effect on cell-mediated immune responses.ResultsExamining MHCI haplotype variation in Atlantic salmon using transcriptome and genome resources we found little evidence for polymorphism in antigen processing genes closely linked to the classical MHCIa genes. Looking at other genes involved in MHCI assembly and antigen processing we found retention of functional gene duplicates originating from the second vertebrate genome duplication event providing cyprinids, salmonids, and neoteleosts with the potential of several different peptide-loading complexes. One of these gene duplications has also been retained in the tetrapod lineage with orthologs in frogs, birds and opossum.ConclusionWe postulate that the unique salmonid whole genome duplication (SGD) is responsible for eliminating haplotypic content in the paralog MHCIa regions possibly due to frequent recombination and reorganization events at early stages after the SGD. In return, multiple rounds of whole genome duplications has provided Atlantic salmon, other teleosts and even lower vertebrates with alternative peptide loading complexes. How this affects antigen presentation remains to be established.

| S-EPMC5824609 | biostudies-literature

Crystal structures of lysophospholipid-bound MHC class I molecules.

Project description:Newly synthesized major histocompatibility complex (MHC) class I proteins are stabilized in the endoplasmic reticulum (ER) by binding 8-10-mer-long self-peptide antigens that are provided by transporter associated with antigen processing (TAP). These MHC class I:peptide complexes then exit the ER and reach the plasma membrane, serving to sustain the steady-state MHC class I expression on the cell surface. A novel subset of MHC class I molecules that preferentially bind lipid-containing ligands rather than conventional peptides was recently identified. The primate classical MHC class I allomorphs, Mamu-B*098 and Mamu-B*05104, are capable of binding the N-myristoylated 5-mer (C14-Gly-Gly-Ala-Ile-Ser) or 4-mer (C14-Gly-Gly-Ala-Ile) lipopeptides derived from the N-myristoylated SIV Nef protein, respectively, and of activating lipopeptide antigen-specific cytotoxic T lymphocytes. We herein demonstrate that Mamu-B*098 samples lysophosphatidylethanolamine and lysophosphatidylcholine containing up to a C20 fatty acid in the ER. The X-ray crystal structures of Mamu-B*098 and Mamu-B*05104 complexed with lysophospholipids at high resolution revealed that the B and D pockets in the antigen-binding grooves of these MHC class I molecules accommodate these lipids through a monoacylglycerol moiety. Consistent with the capacity to bind cellular lipid ligands, these two MHC class I molecules did not require TAP function for cell-surface expression. Collectively, these results indicate that peptide- and lipopeptide-presenting MHC class I subsets use distinct sources of endogenous ligands.

| S-EPMC7242709 | biostudies-literature

Crystal structures of MHC class I complexes reveal the elusive intermediate conformations explored during peptide editing.

Project description:Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between numerous conformational states and these motions support peptide sampling. To date, MHC I intermediates are largely uncharacterized experimentally and remain elusive. Here, we present x-ray crystal structures of HLA-B8 loaded with 20mer peptides that show pronounced distortions at the N-terminus of the groove. Long stretches of N-terminal amino acid residues are missing in the electron density maps creating an open-ended groove. Our structures also reveal highly unusual features in MHC I-peptide interaction at the N-terminus of the groove. Molecular dynamics simulations indicate that the complexes have varying degrees of conformational flexibility in a manner consistent with the structures. We suggest that our structures have captured the remarkable molecular dynamics of MHC I-peptide interaction. The visualization of peptide-dependent conformational motions in MHC I is a major step forward in our conceptual understanding of dynamics in high-affinity peptide selection.

| S-EPMC10439229 | biostudies-literature

Dipeptides catalyze rapid peptide exchange on MHC class I molecules.

Project description:Peptide ligand selection by MHC class I molecules, which occurs by iterative optimization, is the centerpiece of immunodominance in antiviral and antitumor immune responses. For its understanding, the molecular mechanisms of peptide binding and dissociation by class I molecules must be elucidated. To this end, we have investigated dipeptides that bind to the F pocket of class I molecules. We find that they accelerate the dissociation of prebound peptides of both low and high affinity, suggesting a mechanism of action for the peptide-exchange chaperone tapasin. Peptide exchange on class I molecules also has practical uses in epitope discovery and T-cell monitoring.

| S-EPMC4291614 | biostudies-literature

Generation of MHC class II-peptide ligands for CD4 T-cell allorecognition of MHC class II molecules.

Project description:Purpose of reviewThe molecular and cellular mechanisms that underlie allorecognition of MHC class II molecules have been the subject of much debate and experimentation in recent decades. In this review, we discuss several aspects of MHC class II structure, peptide acquisition and TcR-MHC-peptide interactions that have particular relevance to recognition of cells bearing allogeneic class II molecules.Recent findingsFirst, MHC polymorphism is heavily biased toward those amino acids that influence stable peptide binding by MHC class II. Second, the peptide repertoire presented by class II molecules is highly diverse and can be edited substantially by the molecular catalyst HLA-DM and by tissue-specific expression of HLA-DO, stress and cytokines. Third, T-cell receptor docking onto MHC peptide consistently involves substantial contacts with the bound peptide in the MHC class II molecule. Finally, there is increasing evidence that T-cell recognition of MHC is, in part, germline encoded through T-cell-receptor V region contacts with MHC class II alpha helices.SummaryTogether, these conclusions support the view that allorecognition of MHC class II molecules is likely to parallel key aspects of conventional CD4 T-cell recognition, with allele-dependent variation in peptide representation accounting in large part for the high precursor frequency of alloreactive CD4 T cells.

| S-EPMC3085167 | biostudies-literature

Direct peptide-regulatable interactions between MHC class I molecules and tapasin.

Project description:Tapasin (Tpn) has been implicated in multiple steps of the MHC class I assembly pathway, but the mechanisms of function remain incompletely understood. Using purified proteins, we could demonstrate direct binding of Tpn to peptide-deficient forms of MHC class I molecules at physiological temperatures. Tpn also bound to M10.5, a pheromone receptor-associated MHC molecule that has an open and empty groove and that shares significant sequence identity with class I sequences. Two types of MHC class I-Tpn complexes were detectable in vitro depending on the input proteins; those depleted in beta(2)m, and those containing beta(2)m. Both were competent for subsequent assembly with peptides, but the latter complexes assembled more rapidly. Thus, the assembly rate of Tpn-associated class I was determined by the conditions under which Tpn-MHC class I complexes were induced. Peptide loading of class I inhibited Tpn-class I-binding interactions, and peptide-depletion enhanced binding. In combination with beta(2)m, certain peptides induced efficient dissociation of preformed Tpn-class I complexes. Together, these studies demonstrate direct Tpn-MHC class I interactions and preferential binding of empty MHC class I by Tpn, and that the Tpn-class I interaction is regulated by both beta(2)m and peptide. In cells, Tpn is likely to be a direct mediator of peptide-regulated binding and release of MHC class I from the TAP complex.

| S-EPMC1838733 | biostudies-literature

Dipeptides promote folding and peptide binding of MHC class I molecules.

Project description:MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2K(b) into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-2K(b) and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide.

| S-EPMC3780906 | biostudies-literature

Functional recombinant MHC class II molecules and high-throughput peptide-binding assays.

Project description:BackgroundMolecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged.ResultsWe generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately.ConclusionWe have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.

| S-EPMC2690590 | biostudies-literature

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