Secondary Anchor Residues Research Articles

Two distinct HLA-A*0101-specific submotifs illustrate alternative peptide binding modes.

Previous studies have defined two different peptide binding motifs specific for HLA-A*0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A*0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A*0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE3 submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A*0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A*0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A*0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A*0101-restricted peptide epitopes.

Variation in H-2Kk peptide motif revealed by sequencing naturally processed peptides from T-cell hybridoma class I molecules

Class I major histocompatibility complex (MHC) molecules interact with a diverse array of self and foreign peptides, displaying them on the cell surface and providing an extracellular indication of intracellular invasion. The most clearly defined role for these class I/peptide complexes is to cause effector responses upon binding to antigen-specific receptors of cytotoxic T cells. We have characterized the mouse thymoma/rat V beta 8.2+ T-cell hybridoma C14/BW12-12A1 by fluorescence-activated cell sorting analysis and have used immunoaffinity chromatography to purify class I molecules from these cells. The peptides bound to the class I molecules were fractionated by high-performance liquid chromatography and sequenced. Self-peptide mixtures eluted from the mouse H-2Kk class I allele revealed a dominant primary sequence motif, with a carboxyl-terminal residue that appeared to be invariantly valine and a secondary or auxiliary anchor residue at position 2 that could be either glutamate or proline. The majority of naturally processed peptide ligands appeared to be octamers. Although peptides eluted off H-2Kk molecules from tissue derived from a number of different inbred mouse strains also appeared to be octamers, others have reported that isoleucine is the dominant carboxyl-terminal residue. Thus, different cell types displayed distinct differences in naturally processed peptides bound by the same class I alleles. The variation in naturally processed peptides loaded onto the same class I allele most likely reflects the nature of the pool of peptides within the cell available for loading class I molecules.

Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding.

Considerable interest has focused on understanding how major histocompatibility complex (MHC) specificity is generated and characterizing the specificity of MHC molecules with the ultimate goal being to predict peptide binding. We have used a strategy where all possible peptides of a particular size are distributed into positional scanning combinatorial peptide libraries (PSCPL) to develop a highly efficient, universal and unbiased approach to address MHC specificity. The PSCPL approach appeared qualitatively and quantitatively superior to other currently used strategies. The average effect of any amino acid in each position was quantitated, allowing a detailed description of extended peptide binding motifs including primary and secondary anchor residues. It also identified disfavored residues which were found to be surprisingly important in shaping MHC class I specificity. Assuming that MHC class I specificity is the result of largely independently acting subsites, the binding of unknown peptides could be predicted. Conversely, this argues that MHC class I specificities consist of an array of subspecificities acting in a combinatorial mode.

T cell recognition of Tn-glycosylated peptide antigens.

The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attached at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67-76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.

Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules.

Abstract The binding capacity of large sets of peptides corresponding to naturally occurring sequences and carrying previously defined A24-specific motifs was analyzed. It was found that only a minority (9-25%) of the motif-carrying peptides bound the relevant HLA-A molecule with good affinity (IC 50% < or = 50 nM), while the majority of them bound only weakly or not at all (IC 50% > or = 500 nM). By correlating the presence of specific residue types at each position along the peptide sequence with average binding affinity, the prominent influence of specific secondary interactions (secondary anchor residues) was revealed. Moreover, secondary interactions appeared to be size-dependent in that the specific effects detected differed in 9-mer and 10-mer peptide sets. Based on these observations, A24-specific refined motifs were also established for both 9-mer and 10-mer ligands, and their merit was verified by testing the binding capacity of independent sets of synthetic peptides. Such refined motifs should facilitate accurate prediction of potential A24-restricted peptide epitopes. It was also noted that certain crucial secondary interactions appear to be remarkably similar in the case of A24 and other HLA-A molecules previously analyzed (A*0201, A3, A11, and others). This may reflect contributions to binding affinity of relatively invariant residues located within the polymorphic pockets of the HLA binding groove.

Augmentation of the affinity of HLA class I‐binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

A direct binding assay has been used to investigate the effect of the secondary anchor residues on peptide binding to class I proteins of the major histocompatibility complex. Based on predictions from a previous chemometric approach, synthetic peptide analogues containing unnatural amino acids were synthesized and tested for B*2705 binding. Hydrophobic unnatural amino acids such as alpha-naphthyl- and cyclohexyl-alanine were found to be excellent substituents in the P3 secondary anchor position giving peptides with very high B*2705-binding affinity. The binding to B*2705 of peptides optimized for their secondary anchor residues, but lacking one of the P2 or P9 primary anchor residues was also investigated. Most such peptides did not bind, but one peptide, lacking the P2 Arg residue generally considered essential for binding to all B27 subtypes, was found to bind quite strongly. These findings demonstrate that peptide binding to class I proteins is due to a combination of all the anchor residues, which may be occupied also by unnatural amino acids-a necessary step towards the development of peptidic or non-peptidic antagonists for immunomodulation.

Molecular dynamics and structure-based drug design for predicting non-natural nonapeptide binding to a class I MHC protein.

Starting from the known three-dimensional structure of the class I major histocompatibility complex-encoded HLA-B*2705 protein, three non-natural nonapeptides were designed to fit optimally the HLA-B*2705-binding groove. The optimization was performed using structure-based drug design methods and the fact that all the possible interactions of the secondary anchor residue (position 3) with its human leukocyte antigen-binding pocket (pocket D) in nature are not entirely utilized. 150 ps molecular-dynamics (MD) simulation in water was employed to study the stability of the bimolecular complexes with three non-natural peptides (P3 = homophenylalanine, beta-naphthylalanine, alpha-naphthylalanine) as well as with the two natural homologues (P3 = Gly, Leu). Various structural and dynamical properties (atomic fluctuations, solvent-accessible surface areas, peptide Calpha-atom positions) of the simulated bimolecular complexes were used to compare the three non-natural with the two natural ligands. Since the various molecular properties have been shown previously to be related to the binding affinity of nonapeptide ligands to the major histocompatibility complex (MHC) HLA-B*2705 protein, the MD data predict a rather higher stability of MHC-ligand complexes with the three non-natural peptides, suggesting that the unnatural peptides studied show an enhanced binding affinity to the HLA-B*2705 protein. These results are in agreement with the experimental values of a semi-quantitative in vitro assembly assay, performed on the five nonapeptides (P3 = Gly, Leu, homophenylalanine, beta-naphthylalanine, alpha-naphthylalanine), which shows their ability to stabilize the native conformation of the HLA-B*2705 heavy chain and also shows that the three non-natural ligands bind with higher affinity (0.5 micro M) to the MHC protein than the two natural homologues (40 micro M). Thus, this study demonstrates that structural information combined with rational design and molecular-dynamics simulations can illustrate and predict MHC binding and potential T-cell epitope properties as well as contribute to the design of new non-peptidic MHC inhibitors that may be useful for the selective immunotherapy of autoimmune diseases to which HLA alleles are directly associated.

Overlap in the repertoires of peptides bound in vivo by a group of related class I HLA-B allotypes

Polymorphism among class I molecules of the major histocompatibility complex (MHC) confers allotypic specificity on the peptides that these molecules bind and present to cytotoxic T lymphocytes. Evolution of new human HLA class I alleles usually involves gene recombination events that replace a segment of one allele with the homologous region of another. In this study, the impact of these evolutionary changes has been assessed by comparison of the peptide-binding specificities of six related HLA-B allotypes. Endogenous peptides bound by HLA-B*5401, HLA-B*5501, HLA-B*5502, HLA-B*5601, HLA-B*6701 and HLA-B*0702 were characterized. Despite differing by 1-9 of the amino-acid residues comprising their peptide-binding sites, all these allotypes share a dominant preference for peptides that have proline at position 2. Polymorphism results in differing selection of carboxy-terminal and secondary anchor residues, but the peptide-binding specificities are sufficiently similar that there is overlap in the repertoires of peptides bound by these allotypes. Complete sequence determination of individual peptides revealed four that could be isolated from two or more allotypes. Members of the closely related HLA-B22 family--HLA-B*5401, HLA-B*5501, HLA-B*5502 and HLA-B*5601--show only minor differences in their peptide-binding specificities. This marked similarity is reflected at the functional level, as alloreactive cytotoxic T lymphocytes generated against HLA-B*5401 and HLA-B*5501 exhibited cross-reactive recognition. The isolation of identical endogenously bound peptides from six HLA-B allotypes demonstrates overlap in the repertoires of peptides bound in vivo by different allotypes. We speculate that the shared preference for binding peptides with proline at position 2 reflects a selective pressure to retain this specificity, which may be based upon peptide availability in vivo. Characterization of the overlap between the repertoires of peptides bound by HLA-B allotypes could simplify the development of peptide-based vaccines that are targeted to cytotoxic T cells, as single peptides would be effective for humans of different HLA types.

The importance of secondary anchor residue motifs of HLA class I proteins: A chemometric approach

In this paper we report a chemometric approach to Quantitative Structure-Activity Relationship (QSAR) analysis applied to a study of the binding of peptides to Major Histocompatibility Complex (MHC) class I proteins. Peptides which possess the known primary anchor residue motif for HLA-B27 binding do not necessarily bind to HLA-B27 proteins. Secondary anchor residues are also involved, but it is not yet clear which amino acids are required or in which positions. A classic approach to this problem would be to synthesize multiple peptides each varying by a single amino acid from a starting peptide, and test them for their binding properties. Not only is this approach inefficient, but it is essentially unable to provide information about possible mutual interactions of amino acid residues in different positions. Using a statistical design to select the most informative compounds to use in the QSAR study, it was possible to analyse the effects on HLA-B27 peptide binding of different amino acids in four positions by means of only nine peptides. The relative binding activity of these peptides could then be modeled mathematically to provide information about the relative contribution of each of the four positions and to suggest a new peptide with high binding affinity. Our results demonstrate the usefulness of the chemometric strategy for studying peptides of interest in molecular immunology.

Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules

The functional determinants of histocompatibility leukocyte antigen (HLA)-A2.1-peptide interactions have been detailed by the use of quantitative molecular binding assays and a chemically synthesized library of naturally occurring epitopes. The importance of hydrophobic anchor residues in position 2 and the C-terminus was confirmed. These anchors are necessary, but not sufficient, for high affinity binding, as the predictions based solely on these anchors are only about 30% accurate. Prominent roles for several other positions (1, 3, and 7) were also demonstrated. The location of these residues within the peptides matches secondary A2.1 pockets previously demonstrated by X-ray crystallography. From a functional standpoint, similar dominant negative effects on binding were observed for charged residues in both nonamers and decamers, while positive effects differed between nonamers and decamers. An extended motif taking into account secondary anchors increased the predictability of A2.1-binding epitopes to a level of 70%, underscoring the practical usefulness of extended motifs.