Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide.
ABSTRACT: The human major histocompatibility complex class I antigen HLA-B*2705 binds several sequence-related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross-reactivity of cytotoxic T cells (CTL) against these HLA-B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging-in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross-reactivity of these CTL with a peptide derived from voltage-dependent calcium channel ?1 subunit (pCAC, SRRWRRWNR), in which the C-terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA-B*2705:pCAC complex by X-ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence-related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F-pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA-B*2705 heavy chain near the N-terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg-Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR-directed, HLA-B*2705-restricted CTL to cross-react with HLA-B*2705:pCAC complexes.
Project description:The products of the human leukocyte antigen subtypes HLA-B*2705 and HLA-B*2709 differ only in residue 116 (Asp vs. His) within the peptide binding groove but are differentially associated with the autoimmune disease ankylosing spondylitis (AS); HLA-B*2705 occurs in AS-patients, whereas HLA-B*2709 does not. The subtypes also generate differential T cell repertoires as exemplified by distinct T cell responses against the self-peptide pVIPR (RRKWRRWHL). The crystal structures described here show that pVIPR binds in an unprecedented dual conformation only to HLA-B*2705 molecules. In one binding mode, peptide pArg5 forms a salt bridge to Asp116, connected with drastically different interactions between peptide and heavy chain, contrasting with the second, conventional conformation, which is exclusively found in the case of B*2709. These subtype-dependent differences in pVIPR binding link the emergence of dissimilar T cell repertoires in individuals with HLA-B*2705 or HLA-B*2709 to the buried Asp116/His116 polymorphism and provide novel insights into peptide presentation by major histocompatibility antigens.
Project description:The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P2(1)2(1)2(1). Data sets were collected to 1.60 A (complex with the self-peptide pVIPR) or to 1.90 A (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.
Project description:The human leukocyte antigen (HLA) alleles HLA-B*2704 and HLA-B*2706 show an ethnically restricted distribution and are differentially associated with ankylosing spondylitis, with HLA-B*2706 lacking association with this autoimmune disease. However, the products of the two alleles differ by only two amino acids, at heavy-chain residues 114 (His in HLA-B*2704; Asp in HLA-B*2706) and 116 (Asp in HLA-B*2704; Tyr in HLA-B*2706). Both residues could be involved in contacting amino acids of a bound peptide, suggesting that peptides presented by these subtypes play a role in disease pathogenesis. Two HLA-B*2706-peptide complexes were crystallized using the hanging-drop vapour-diffusion method with PEG as precipitant. Data sets were collected to resolutions of 2.70 A (viral peptide pLMP2, RRRWRRLTV; space group P2(1)2(1)2(1)) and 1.83 A (self-peptide pVIPR, RRKWRRWHL; space group P2(1)). Using HLA-B*2705 complexed with the pGR peptide (RRRWHRWRL) as a search model, unambiguous molecular-replacement solutions were found for both HLA-B*2706 complexes.
Project description:The development of autoimmune disorders is incompletely understood. Inefficient thymic T cell selection against self-peptides presented by major histocompatibility antigens (HLA in humans) may contribute to the emergence of auto-reactive effector cells, and molecular mimicry between foreign and self-peptides could promote T cell cross-reactivity. A pair of class I subtypes, HLA-B2705 and HLA-B2709, have previously been intensely studied, because they are distinguished from each other only by a single amino acid exchange at the floor of the peptide-binding groove, yet are differentially associated with the autoinflammatory disorder ankylosing spondylitis. Using X-ray crystallography in combination with ensemble refinement, we find that the non-disease-associated subtype HLA-B2709, when presenting the self-peptide pGR (RRRWHRWRL), exhibits elevated conformational dynamics, and the complex can also be recognized by T cells. Both features are not observed in case of the sequence-related self-peptide pVIPR (RRKWRRWHL) in complex with this subtype, and T cell cross-reactivity between pGR, pVIPR, and the viral peptide pLMP2 (RRRWRRLTV) is only rarely observed. The disease-associated subtype HLA-B2705, however, exhibits extensive conformational flexibility in case of the three complexes, all of which are also recognized by frequently occurring cross-reactive T cells. A comparison of the structural and dynamic properties of the six HLA-B27 complexes, together with their individual ability to interact with T cells, permits us to correlate the flexibility of HLA-B27 complexes with effector cell reactivity. The results suggest the existence of an inverse relationship between conformational plasticity of peptide-HLA-B27 complexes and the efficiency of negative selection of self-reactive cells within the thymus.
Project description:The existence of cytotoxic T cells (CTL) cross-reacting with the human major histocompatibility antigens HLA-B14 and HLA-B27 suggests that their alloreactivity could be due to presentation of shared peptides in similar binding modes by these molecules. We therefore determined the crystal structures of the subtypes HLA-B*1402, HLA-B*2705, and HLA-B*2709 in complex with a proven self-ligand, pCatA (peptide with the sequence IRAAPPPLF derived from cathepsin A (residues 2-10)), and of HLA-B*1402 in complex with a viral peptide, pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244) of Epstein-Barr virus). Despite the exchange of 18 residues within the binding grooves of HLA-B*1402 and HLA-B*2705 or HLA-B*2709, the pCatA peptide is presented in nearly identical conformations. However, pLMP2 is displayed by HLA-B*1402 in a conformation distinct from those previously found in the two HLA-B27 subtypes. In addition, the complexes of HLA-B*1402 with the two peptides reveal a nonstandard, tetragonal mode of the peptide N terminus anchoring in the binding groove because of the exchange of the common Tyr-171 by His-171 of the HLA-B*1402 heavy chain. This exchange appears also responsible for reduced stability of HLA-B14-peptide complexes in vivo and slow assembly in vitro. The studies with the pCatA peptide uncover that CTL cross-reactive between HLA-B14 and HLA-B27 might primarily recognize the common structural features of the bound peptide, thus neglecting amino acid replacements within the rim of the binding grooves. In contrast, structural alterations between the three complexes with the pLMP2 peptide indicate how heavy chain polymorphisms can influence peptide display and prevent CTL cross-reactivity between HLA-B14 and HLA-B27 antigens.
Project description:The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the ?1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.
Project description:The frequency of HLA-B27 in patients with Ankylosing Spondylitis (AS) is over 85%. There are more than 170 recognized HLA-B27 alleles but the majority of them is not sufficiently represented for genetic association studies. So far only two alleles, the HLA-B*2706 in Asia and the HLA-B*2709 in Sardinia, have not been found to be associated with AS. The highly homogenous genetic structure of the Sardinian population has favored the search of relevant variants for disease-association studies. Moreover, malaria, once endemic in the island, has been shown to have contributed to shape the native population genome affecting the relative allele frequency of relevant genes. In Sardinia, the prevalence of HLA-B*2709, which differs from the strongly AS-associated B*2705 prototype for one amino acid (His/Asp116) in the F pocket of the peptide binding groove, is around 20% of all HLA-B27 alleles. We have previously hypothesized that malaria could have contributed to the establishment of this allele in Sardinia. Based on our recent findings, in this perspective article we speculate that the Endoplasmic Reticulum Amino Peptidases, ERAP1 and 2, associated with AS and involved in antigen presentation, underwent co-selection by malaria. These genes, besides shaping the immunopeptidome of HLA-class I molecules, have other biological functions that could also be involved in the immunosurveillance against malaria.
Project description:Analysis of antigen dissociation provides insight into peptide presentation modes of folded human leukocyte antigen (HLA) molecules, which consist of a heavy chain, beta2-microglobulin (beta2m), and an antigenic peptide. Here we have monitored peptide-HLA interactions and peptide dissociation kinetics of two HLA-B27 subtypes by fluorescence depolarization techniques. A single natural amino-acid substitution distinguishes the HLA-B*2705 subtype that is associated with the autoimmune disease ankylosing spondylitis from the non-disease-associated HLA-B*2709 subtype. Peptides with C-terminal Arg or Lys represent 27% of the natural B*2705 ligands. Our results show that dissociation of a model peptide with a C-terminal Lys (GRFAAAIAK) follows a two-step mechanism. Final peptide release occurs in the second step for both HLA-B27 subtypes. However, thermodynamics and kinetics of peptide-HLA interactions reveal different molecular mechanisms underlying the first step, as indicated by different activation energies of 95+/-8 kJ/mol (HLA-B*2705) and 150+/-10 kJ/mol (HLA-B*2709). In HLA-B*2709, partial peptide dissociation probably precedes fast final peptide release, while in HLA-B*2705 an allosteric mechanism based on long-range interactions between beta2m and the peptide binding groove controls the first step. The resulting peptide presentation mode lasts for days at physiological temperature, and determines the peptide-HLA-B*2705 conformation, which is recognized by cellular ligands such as T-cell receptors.
Project description:HLA-B*27 is strongly associated with an inflammatory autoimmune disorder, the Ankylosing Spondylitis (AS) and plays a protective role in viral infections. The two aspects might be linked. In this work, we compared in B*2705/B*07 positive patients with AS, the CD8+ T cell responses to two immunodominant EBV-derived epitopes restricted for either the HLA-B*27 (pEBNA3C) or the HLA-B*07 (pEBNA3A). We have unexpectedly found that the HLA-B*07-restricted EBNA3A peptide is presented by both the B*0702 and the B*2705 but not by the non AS-associated B*2709, that differs from the AS-associated B*2705 for a single amino acid in the peptide-binding groove (His116Asp). We then analysed 38 B*2705-positive/B*07-negative (31 AS-patients and 7 healthy donors) and 8 B*2709-positive/B*07-negative subjects. EBNA3A-specific CD8+ T lymphocytes were present in 55.3% of the HLA-B*2705 but in none of the B*2709 donors (p=0.0049). TCR ?-chain analysis identified common TCRBV and TCRBJ gene segments and shared CDR3? sequences in pEBNA3A-responsive CTLs of B*2705 carriers, suggesting the existence of a shared TCR repertoire for recognition of the uncanonical B*2705/pEBNA3A complex. These data highlight the plasticity of the AS-associated HLA-B*2705, which presents peptides with suboptimal binding motifs, possibly contributing both to its enhanced capacity to protect against pathogens and to predispose to autoimmunity.
Project description:The human leukocyte antigen HLA-B27 is a strong risk factor for Ankylosing Spondylitis (AS), an immune-mediated disorder affecting axial skeleton and sacroiliac joints. Additionally, evidence exists sustaining a strong protective role for HLA-B27 in viral infections. These two aspects could stem from common molecular mechanisms. Recently, we have found that the HLA-B*2705 presents an EBV epitope (pEBNA3A-RPPIFIRRL), lacking the canonical B27 binding motif but known as immunodominant in the HLA-B7 context of presentation. Notably, 69% of B*2705 carriers, mostly patients with AS, possess B*2705-restricted, pEBNA3A-specific CD8+ T cells. Contrarily, the non-AS-associated B*2709 allele, distinguished from the B*2705 by the single His116Asp polymorphism, is unable to display this peptide and, accordingly, B*2709 healthy subjects do not unleash specific T cell responses. Herein, we investigated whether the reactivity towards pEBNA3A could be a side effect of the recognition of the natural longer peptide (pKEBNA3A) having the classical B27 consensus (KRPPIFIRRL). The stimulation of PBMC from B*2705 positive patients with AS in parallel with both pEBNA3A and pKEBNA3A did not allow to reach an unambiguous conclusion since the differences in the magnitude of the response measured as percentage of IFN?-producing CD8+ T cells were not statistically significant. Interestingly, computational analysis suggested a structural shift of pEBNA3A as well as of pKEBNA3A into the B27 grooves, leaving the A pocket partially unfilled. To our knowledge this is the first report of a viral peptide: HLA-B27 complex recognized by TCRs in spite of a partially empty groove. This implies a rethinking of the actual B27 immunopeptidome crucial for viral immune-surveillance and autoimmunity.