Structural prediction of peptides binding to MHC class I molecules

Peptide binding to class I major histocompatibility complex (MHCI) molecules is a key step in the immune response and the structural details of this interaction are of importance in the design of peptide vaccines. Algorithms based on primary sequence have had success in predicting potential antigenic peptides for MHCI, but such algorithms have limited accuracy and provide no structural information. Here, we present an algorithm, PePSSI (peptide-MHC prediction of structure through solvated interfaces), for the prediction of peptide structure when bound to the MHCI molecule, HLA-A2. The algorithm combines sampling of peptide backbone conformations and flexible movement of MHC side chains and is unique among other prediction algorithms in its incorporation of explicit water molecules at the peptide-MHC interface. In an initial test of the algorithm, PePSSI was used to predict the conformation of eight peptides bound to HLA-A2, for which X-ray data are available. Comparison of the predicted and X-ray conformations of these peptides gave RMSD values between 1.301 and 2.475 A. Binding conformations of 266 peptides with known binding affinities for HLA-A2 were then predicted using PePSSI. Structural analyses of these peptide-HLA-A2 conformations showed that peptide binding affinity is positively correlated with the number of peptide-MHC contacts and negatively correlated with the number of interfacial water molecules. These results are consistent with the relatively hydrophobic binding nature of the HLA-A2 peptide binding interface. In summary, PePSSI is capable of rapid and accurate prediction of peptide-MHC binding conformations, which may in turn allow estimation of MHCI-peptide binding affinity.     

Crystal structures of HLA-A*0201 complexed with antigenic peptides with either the amino- or carboxyl-terminal group substituted by a methyl group

The crystal structures of class I major histocompatibility complex (MHC) molecules complexed with antigenic peptides revealed a network of hydrogen bonds between the charged amino- and carboxyl-termini of the peptides and conserved MHC residues at both ends of the peptide binding site. These interactions were shown to contribute substantially to the stability of class I MHC/peptide complexes by thermal denaturation studies using synthetic peptides in which either the amino- or carboxyl-terminal group is substituted by a methyl group. Here we report crystal structures of HLA-A*0201 complexed with these terminally modified synthetic peptides showing that they adopt the same bound conformation as antigenic peptides. A number of variations in peptide conformation were observed for the terminally modified peptides, including in one case, a large conformational difference in four central peptide residues that is apparently caused by the lattice contact. This is reminiscent of the way binding a T-cell receptor changed the conformation of central residues of an MHC-bound peptide. The structures determined identify which conserved hydrogen bonds are eliminated in terminally substituted peptides and suggest an increased energetic importance of the interactions at the peptide termini for MHC-peptide stability. Proteins 33:97–106, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.     

Molecular dynamics study of a complex between the human histocompatibility antigen HLA-A2 and the IMP58-66 nonapeptide from influenza virus matrix protein.

The structure of the influenza-virus-matrix-protein (IMP) 58-66 nonapeptide, bound to the major-histocompatibility-complex-encoded human leukocyte antigen (HLA) A2 protein was studied by molecular dynamics simulation. Starting from the extra electron density map of peptides co-crystallized with HLA-A2, the nonapeptide IMP58-66 was docked residue by residue in the protein binding cleft. The complex was simulated for 100 ps in a shell of 1372 water molecules. The averaged simulated HLA-A2 conformation was found to be similar to the crystal structure (0.182 nm RMS deviation, for the backbone atoms of the alpha 1-alpha 2 domain). Nine out of the 14 hydrogen bonds observed in the antigen-binding site were reproduced in the simulation. The IMP58-66 peptide exhibits an extended conformation with kinks at positions 3 and 5. The side chains of residues 2, 3 and 9 develop van der Waals' interactions with hydrophobic pockets of HLA-A2, corresponding to polymorphic residues of the major-histocompatibility-complex-encoded proteins. Both the N-terminus and C-terminus of the nonapeptide were anchored in the antigen-binding groove by hydrogen bonds with conserved amino acids. The N-terminus was more flexible and contacts four HLA-A2 conserved tyrosines (Tyr7, Tyr59, Tyr159 and Tyr171) and Glu63 by direct or water-relayed hydrogen bonds. Water intercalation occurred only around the N-terminus of the peptide, the C-terminal carboxylate forming strong hydrogen bonds with polar residues (Tyr84 and Thr143) and a salt bridge with Lys146 all over the molecular dynamics simulation. This model is fully compatible with the recently published crystal structure of the HLA-B27 protein, complexed by a mixture of self nonapeptides.     

Structural and immunological reactivity of the principal neutralizing determinant V3 of glycoprotein gp 120 of HIV-1

The variable domain V3 in the outer glycoprotein gp 120 of HIV-1 is a highly important region with respect to immune response during the course of viral infection. Neutralizing antibodies are produced against this domain; in addition, it has been shown to be a functionally active epitope for T helper and cytotoxic T cells. The high degree of amino acid variability in individual HIV-isolates, however, limits the use of the V3-domain in approaches to vaccine development. In order to characterize the residues important for antibody interaction and binding to MHC class I proteins, we constructed a consensus sequence of the V3-domain with broad reactivity [1] and used synthetic peptides derived from this consensus with individual residues altered to alanine. These peptides were used as antigens in ELISA tests to define the amino acids which are important for binding to human and rabbit/anti-peptide immunoglobulins. In addition, we used these alanine-derived peptides in interaction studies with human HLA-A2.1 and mouse H-2D^d by testing their capacity to stabilize the respective MHC class I protein complexes on the surface of mutant cell lines T2 and RMA-S transfected with D^d gene. The experimental tests allowed us to define individual residues involved in antibody and MHC-protein interaction, respectively. In a further approach, we used those results to design interaction models with HLA-A2.1 and H-2D^d. Therefore, a structural model for H-2D^d was built that exhibits an overall similar conformation to the parental crystal structure of HLA-A2.1. The resulting interaction models show V3-peptide bound in an extended β-conformation with a bulge in its centre for both H-2D^d and HLA-A2.1 complexes. The N- and C-termini of V3 peptide reside in conserved pockets within both MHC-proteins. Anchoring residues could be determined that are crucial for the binding of the respective MHC class I haplotype. The cross-reactivity of V3-peptide in enhancing the expression of two different MHC class I molecules (H-2D^d and HLA-A2.1) is shown to be based on similar peptide binding that induces an almost identical peptide conformation.     

An extensive region of an MHC class I alpha 2 domain loop influences interaction with the assembly complex.

Presentation of antigenic peptides to CTLs at the cell surface first requires assembly of MHC class I with peptide and beta 2-microglobulin in the endoplasmic reticulum. This process involves an assembly complex of several proteins, including TAP, tapasin, and calreticulin, all of which associate specifically with the beta 2-microglobulin-assembled, open form of the class I heavy chain. To better comprehend at a molecular level the regulation of class I assembly, we have assessed the influence of multiple individual amino acid substitutions in the MHC class I alpha 2 domain on interaction with TAP, tapasin, and calreticulin. In this report, we present evidence indicating that many residues surrounding position 134 in H-2Ld influence interaction with assembly complex components. Most mutations decreased association, but one (LdK131D) strongly increased it. The Ld mutants, with the exception of LdK131D, exhibited characteristics suggesting suboptimal intracellular peptide loading, similar to the phenotype of Ld expressed in a tapasin-deficient cell line. Notably, K131D was less peptide inducible than wild-type Ld, which is consistent with its unusually strong association with the endoplasmic reticulum assembly complex.     

The H-2Kk MHC peptide-binding groove anchors the backbone of an octameric antigenic peptide in an unprecedented mode

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2(b) and H-2(d) haplotypes. In contrast, there is a dearth of structural data regarding H-2(k)-encoded molecules. Therefore, the structures of H-2K(k) complexed to an octameric peptide from influenza A virus (HA(259-266)) and to a nonameric peptide from SV40 (SV40(560-568)) have been determined by x-ray crystallography at 2.5 and 3.0 A resolutions, respectively. The structure of the H-2K(k)-HA(259-266) complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2K(k) groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2K(k) peptide-binding groove. As a result, the Calpha atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2K(b)-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2K(k), the backbone of the SV40(560-568) nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2L(d). This structural convergence occurs despite the totally different architectures of the H-2L(d) and H-2K(k) peptide-binding grooves. Therefore, these two H-2K(k)-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.     

Folding study of an Aib-rich peptide in DMSO by molecular dynamics simulations.

To evaluate the ability of molecular dynamics (MD) simulations using atomic force-fields to correctly predict stable folded conformations of a peptide in solution, we show results from MD simulations of the reversible folding of an octapeptide rich in alpha-aminoisobutyric acid (2-amino-2-methyl-propanoic acid, Aib) solvated in di-methyl-sulfoxide (DMSO). This solvent generally prevents the formation of secondary structure, whereas Aib-rich peptides show a high propensity to form secondary structural elements, in particular 3(10)- and alpha-helical structures. Aib is, moreover, achiral, so that Aib-rich peptides can form left- or right-handed helices depending on the overall composition of the peptide, the temperature, and the solvation conditions. This makes the system an interesting case to study the ensembles of peptide conformations as a function of temperature by MD simulation. Simulations involving the folding and unfolding of the peptide were performed starting from two initial structures, a right-handed alpha-helical structure and an extended structure, at three temperatures, 298 K, 340 K, and 380 K, and the results are compared with experimental nuclear magnetic resonance (NMR) data measured at 298 K and 340 K. The simulations generally reproduce the available experimental nuclear Overhauser effect (NOE) data, even when a wide range of conformations is sampled at each temperature. The importance of adequate statistical sampling in order to reliably interpret the experimental data is discussed.     

Comparison of the conformation and electrostatic surface properties of magainin peptides bound to sodium dodecyl sulfate and dodecylphosphocholine micelles

The role played by noncovalent interactions in inducing a stable secondary structure onto the sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelle-bound conformations of (Ala(8,13,18))magainin 2 amide and the DPC micelle bound conformation of magainin 1 were determined. Two-dimensional NMR and molecular modeling investigations indicated that (Ala(8,13,18))magainin 2 amide bound to DPC micelles adopts a alpha-helical secondary structure involving residues 2-16. The four C-terminal residues converge to a lose beta-turn structure. (Ala(8,13,18))magainin 2 amide bound to SDS miscelles adopts a alpha-helical secondary structure involving residues 7-18. The C- and N-terminal residues exhibited a great deal of conformational flexibility. Magainin 1 bound to DPC micelles adopts a alpha-helical secondary structure involving residues 4-19. The C-terminal residues converge to a lose beta-turn structure. The results of this investigation indicate hydrophobic interactions are the major contributors to stabilizing the induced helical structure of the micelle-bound peptides. Electrostatic interactions between the polar head groups of the micelle and the cationic side chains of the peptides define the positions along the peptide backbone where the helical structures begin and end.     

Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2(+) patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654-662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.     

Homology modeling and molecular dynamics simulations of MUC1-9/H-2Kb complex suggest novel binding interactions

Human MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K^b. The structure of MUC1-9 in complex with H-2K^b has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K^b complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide’s central region, partially characterized as a β-turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K^b.     

Positive selection is limited by available peptide-dependent MHC conformations

Recent data suggest that the diversity of self peptides presented in the thymus during development contributes to positive selection of a diverse T cell repertoire. We sought to determine whether a previously defined "hole in the immunological repertoire" could be explained by the absence of an appropriate selecting self peptide. The repertoire defect in question is the inability of bm8 mice to make an H-2K-restricted response to OVA. Like other OVA-specific, H-2K-restricted receptors, OT-I-transgenic T cells are not positively selected in bm8 mice. Using criteria we had previously established for identifying positive selection ligands, we found peptides that could restore positive selection of OT-I thymocytes in bm8 mice. Thus, the T cell repertoire can be limited by a requirement for specific self peptides during development. Data with MHC-specific Abs suggested that peptides might be able to force MHC residues to adopt different conformations in Kb vs Kbm8. This shows that peptides can potentially contribute to ligand diversity both directly (via variability in the solvent-exposed side chains) and indirectly (through their effect on the MHC conformation). Our data support a model where self peptide diversity allows selection of T cells specific for a broad range of MHC conformations.     

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.     

Strong conformational propensities enhance T cell antigenicity

The ability to predict T cell antigenic peptides would have important implications for the development of artificial vaccines. As a first step towards prediction, this report uses a new statistical technique to discover and evaluate peptide properties correlating with T cell antigenicity. This technique employs Monte Carlo computer experiments and is applicable to many problems involving protein or DNA. The technique is used to evaluate the contribution of various peptide properties to helper T cell antigenicity. The properties investigated include amphipathicities (alpha and beta), conformational propensities (alpha, beta, turn and coil), and the correlates of alpha-helices, such as the absence of helix-breakers and the positioning of the residues which stabilize alpha-helical dipoles. We also investigate segmental amphipathicity. (A peptide has this property when it contains at least two disjoint subpeptides, one hydrophobic, one hydrophilic.) Statistical correlations and stratifications assessed independent contributions to T cell antigenicity. The findings presented here have important implications for the manufacture of peptide vaccines. These implications are as follows: if possible, peptide vaccines should probably be those protein segments which have a propensity to form amphipathic alpha-helices, which do not have regions with a propensity to coil conformations, and which have a lysine at their COOH-terminus. The last two observations are of particular use in manufacturing peptides vaccines: they indicate where the synthetic peptides should be terminated. These implications are supported by the findings given below. The significances (p values) support the following statistical generalites about antigenic conformations: most helper T cell antigenic sites are amphipathic alpha-helices; alpha-helical amphipathicity and propensity to an alpha-helical conformation contribute independently to T cell antigenicity; there is evidence that some T cell antigenic sites are beta conformations instead of alpha-helices; T cell antigenic sites avoid random coiled conformations; and T cell antigenic sites are usually not segmentally amphipathic. alpha-Helical amphipathicity was significant, but segmental amphipathicity was not. This has implications for the dimensions of the structure interacting with the hydrophobic portion of an amphipathic T cell antigenic site. Lysines are unusually frequent at the COOH-terminal of T cell antigenic sites, even after accounting for tryptic digests. These lysines can stabilize alpha-helical peptides by a favorable interaction with alpha-helical dipoles.(ABSTRACT TRUNCATED AT 400 WORDS)     

A naturally occurring peptide recognized by alloreactive CD8+ cytotoxic T lymphocytes in association with a class I MHC protein.

The antigenic structures that initiate T cell responses to foreign (allogeneic) cells have long attracted considerable interest. We have purified and sequenced a peptide from mouse spleen that is recognized in association with the class I MHC protein H-2Ld by 2C, an alloreactive CD8+ T cell clone. The peptide (LSP-FPFDL) greatly enhances the susceptibility of Ld+ cells to lysis by 2C, and this activity is completely blocked by a clonotypic antibody against the 2C T cell receptor. Thus, this study characterizes the naturally occurring peptide moiety of an MHC-I/peptide complex recognized by alloreactive CD8+ T cells. The peptide, which occurs in the thymus of MHC-disparate mice, can be used to study T cell development in mice expressing transgenes for the 2C T cell receptor.     

Exploiting the Peptide — MHC Water Interface in the Computer-Aided Design of Non-Natural Peptides that Bind to the Class I MHC Molecule HLA-A2

Abstract

Class I major histocompatibility complex (MHC) molecules bind peptides derived from intra-cellular proteins and present them to cytotoxic T cells. Certain human immunological diseases are associated with errors in this process. Here we describe an approach to the design of non-natural peptides that could potentially interfere with peptide presentation associated with autoimmune diseases. We have shown previously that the interaction of the peptide GILGFVFTL with the MHC molecule HLA-A2 is mediated by a network of water molecules. In principle, the addition of hydroxyl groups to the peptide could allow for an enhanced interaction of the modified peptide with this water network. Here we illustrate this approach using a peptide having the non-natural amino acid homoserine at position 3, GIhSGFVFTL, and also peptides in which the Cα(F5)—CO—NH1—Cα(V6) peptide bond is replaced by an ether. Cα(F5)—CH(X)—O—Cα(V6), to give the non-natural peptide GILGF—CH(X)—O—VFTL, where X = CH₂OH or CH₃. In a 200 ps solvated molecular dynamics simulation of the HLA-A2 complexes of each peptide for GIhSGFVFTL and GILGF—CH(CH₂OH)—O—VFTL the peptide conformation remained essentially unchanged from that of GILGFVFTL in the X-ray structure of its complex with HLA-A2. In contrast, for GILGF—CH(CH₃)—O—VFTL the peptide conformation deviated from the X-ray conformation, indicating the importance of the hydroxyl group.     

Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 beta of an MHC class I-restricted TCR

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.     

Structural basis for degenerate recognition of natural HIV peptide variants by cytotoxic lymphocytes

It is well established that even small changes in amino acid side chains of antigenic peptide bound to major histocompatibility complex (MHC) protein may completely abrogate recognition of the peptide-MHC (pMHC) complex by the T cell receptor (TCR). Often, however, several nonconservative substitutions in the peptide antigen are accommodated and do not impair its recognition by TCR. For example, a preponderance of natural sequence variants of the human immunodeficiency virus p17 Gag-derived peptide SLYNTVATL (SL9) are recognized by cytotoxic T lymphocytes, which implies that interactions with SL9 variants are degenerate both with respect to the class I MHC molecule and with respect to TCR. Here we study the molecular basis for this degenerate recognition of SL9 variants. We show that several SL9 variants bind comparably well to soluble HLA-A2 and to a particular soluble TCR and that these variants are active in the cognate cytotoxicity assay. Natural SL9 variation is restricted by its context in the HIV p17 matrix protein. High resolution crystal structures of seven selected SL9 variants bound to HLA-A2 all have remarkably similar peptide conformations and side-chain dispositions outside sites of substitution. This preservation of the peptide conformation despite epitope variations suggests a mechanism for the observed degeneracy in pMHC recognition by TCR and may contribute to the persistence of SL9-mediated immune responses in chronically infected individuals.     

NMR studies of an oligoproline-containing peptide analogue that binds specifically to the H-2Kd histocompatibility molecule

T lymphocytes expressing variable cell surface antigen receptors recognize "processed" forms of antigen, presented on the surface of other cells by molecules of the major histocompatibility complex (MHC). Naturally processed antigenic peptides can be replaced by synthetic ones. The synthetic peptide AYPPPPPTLA (P5) is an active competitor to the antigenic peptide HLA A24 170-182 (sequence RYLENGKETLQRA) that is recognized by A24 specific T cells in association with the H-2Kd class I MHC molecule. In P5 the five prolines were designed to play the role of a rigid spacer between the residue Y and the T-L unit, so as to mimic the role of Y171, T178, and L179 in the HLA A24 antigenic peptide, since these residues have proven to be the most important with respect to the binding of the HLA A24 peptide with the H-2Kd MHC molecule. Nuclear magnetic resonance studies allow us to demonstrate that in aqueous solution P5 adopts at least three long-lived conformations that can be classified with respect to the Y2-P3-P4 amide bonds as trans-trans, cis-trans, and cis-cis. Among these, the trans-trans form is present in 67% of the molecules while the two others share the remaining 33%.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.