Conserved MHC class I peptide binding motif between humans and rhesus macaques

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.     

A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens

As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I phospholigands is challenging as the molecular determinants of the presentation of such post-translationally modified peptides are not fully understood. Here, we employed a peptidomic workflow to identify 256 unique phosphorylated ligands associated with HLA-B*40, -B*27, -B*39, or -B*07. Remarkably, these phosphopeptides showed similar molecular features. Besides the specific anchor motifs imposed by the binding groove of each allotype, the predominance of phosphorylation at peptide position 4 (P4) became strikingly evident, as was the enrichment of basic residues at P1. To determine the structural basis of this observation, we carried out a series of peptide binding assays and solved the crystal structures of HLA-B*40 in complex with a phosphorylated ligand or its nonphosphorylated counterpart. Overall, our data provide a clear explanation to the common motif found in the phosphopeptidomes associated to different HLA-B molecules. The high prevalence of phosphorylation at P4 is dictated by the presence of the conserved residue Arg62 in the heavy chain, a structural feature shared by most HLA-B alleles. In contrast, the preference for basic residues at P1 is allotype-dependent and might be linked to the structure of the A pocket. This molecular understanding of the presentation of phosphopeptides by HLA-B molecules provides a base for the improved prediction and identification of phosphorylated neo-antigens, as potentially used for cancer immunotherapy.     

Position 45 influences the peptide binding motif of HLA-B*44:08

Position 45 represents a highly polymorphic residue within HLA class I alleles, which contacts the p2 position of bound peptides in 85% of the peptide–HLA structures analyzed, while the neighboring residues 41 and 46 are not involved in peptide binding. To investigate the influence of residue 45 at the functional level, we sequenced peptides eluted from recombinant HLA-B*44:08^{41Ala/45Met/46Ala} molecules and compared their features with known peptides from B*44:02^{41Thr/45Lys/46Glu}. While HLA-B*44:02 has an anchor motif of E at the p2 anchor position, HLA-B*44:08 exhibits Q and L as anchor motif. The 45^Met/Lys polymorphism contributes to the alteration in the peptide-binding motif and provides further evidence that mismatches at position 45 should be considered as nonpermissive in a transplantation setting.     

Amino acid 95 causes strong alteration of peptide position PΩ in HLA-B*41 variants

There have been several attempts over the years to identify positions in the peptide-binding region (PBR) of human leukocyte antigens (HLA) that influence the specificity of bound amino acids (AAs) at each position in the peptide. Originally, six pockets (A-F) were defined by calculating the surface area of the PBR on the crystal structure of HLA-A2 molecules. More recent crystallographic analyses of a variety of HLA alleles have led to broader pocket definitions. In this study, we examined the peptide-binding specificity of HLA-B*41 alleles and compared our results with the available pocket definitions. By generating recombinant HLA-B molecules and studying the eluted peptides by mass spectrometry and pool sequencing, we detected two different POmega peptide motifs within the B*41 group: Leu vs Val/Pro. Specificity was dependent on the presence of Leu (B*4102, B*4103, and B*4104) vs Trp (B*4101, B*4105, and B*4106) at AA position 95 in the HLA molecule, whose impact on POmega has been a subject of controversy in current pocket definitions. In contrast, the Arg97Ser mutation did not affect pocket F binding specificity in B*41 subtypes although residue 97 was previously identified as a modulator of peptide binding for several HLA class I alleles. According to most pocket definitions, this study shows that the Asn80Lys substitution in B*4105 impels the peptide's POmega anchor toward more promiscuity. Our sequencing results of peptides eluted from HLA-B*41 variants demonstrate the limitations of current pocket definitions and underline the need for an extended peptide motif database for improved understanding of peptide-major histocompatibility complex interactions.     

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.     

Identification of novel HLA-B27 ligands derived from polymorphic regions of its own or other class I molecules based on direct generation by 20 S proteasome.

HLA-B27 is strongly associated with ankylosing spondylitis. Natural HLA-B27 ligands derived from polymorphic regions of its own or other class I HLA molecules might be involved in autoimmunity or provide diversity among HLA-B27-bound peptide repertoires from individuals. In particular, an 11-mer spanning HLA-B27 residues 169-179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria. Proteasomal digestion of synthetic substrates demonstrated direct generation of the B27-(169-179) ligand. Cleavage after residue 181 generated a B27-(169-181) 13-mer that was subsequently found as a natural ligand of B*2705 and B*2704. Its binding to HLA-B27 subtypes in vivo correlated better than B27-(169-179) with association to spondyloarthropathy. Proteasomal cleavage generated also a peptide spanning B*2705 residues 150-158. This region is polymorphic among HLA-B27 subtypes and class I HLA antigens. The peptide was a natural B*2704 ligand. Since this subtype differs from B*2705 at residue 152, it was concluded that the ligand arose from HLA-B*3503, synthesized in the cells used as a source for B*2704-bound peptides. Thus, polymorphic HLA-B27 ligands derived from HLA-B27 or other class I molecules are directly produced by the 20 S proteasome in vitro, and this can be used for identification of such ligands in the constitutive HLA-B27-bound peptide pool.     

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

CTL recognition of a bulged viral peptide involves biased TCR selection

MHC class I molecules generally present peptides of 8-10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related alphabeta TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.     

Characteristics of HIV-1 Nef regions containing multiple CD8+ T cell epitopes: wealth of HLA-binding motifs and sensitivity to proteasome degradation

First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90-97, 135-143, 71-81, 77-85, 90-100, 73-82, and 128-137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef(66-100). This fragment was efficiently cleaved, and NH(2)-terminally extended precursors of epitope 71-81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.     

Gorillas with spondyloarthropathies express an MHC class I molecule with only limited sequence similarity to HLA-B27 that binds peptides with arginine at P2

The human MHC class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies (SpAs). HLA-B27-transgenic rodents develop SpAs, implicating HLA-B27 in the etiology of these disorders. Several nonhuman primates, including gorillas, develop signs of SpAs indistinguishable from clinical signs of humans with SpAs. To determine whether SpAs in gorillas have a similar HLA-B27-related etiology, we analyzed the MHC class I molecules expressed in four affected gorillas. Gogo-B01, isolated from three of the animals, has only limited similarity to HLA-B27 at the end of the alpha1 domain. It differs by several residues in the B pocket, including differences at positions 45 and 67. However, the molecular model of Gogo-B*0101 is consistent with a requirement for positively charged residues at the second amino acid of peptides bound by the MHC class I molecule. Indeed, the peptide binding motif and sequence of individual ligands eluted from Gogo-B*0101 demonstrate that, like HLA-B27, this gorilla MHC class I molecule binds peptides with arginine at the second amino acid position of peptides bound by the MHC class I molecule. Furthermore, live cell binding assays show that Gogo-B*0101 can bind HLA-B27 ligands. Therefore, although most gorillas that develop SpAs express an MHC class I molecule with striking differences to HLA-B27, this molecule binds peptides similar to those bound by HLA-B27.     

Residue 81 confers a restricted C-terminal peptide binding motif in HLA-B*44:09

Knowledge about the magnitude of individual polymorphism is a critical part in understanding the complexity of comprehensive mismatching. HLA-B*44:09 differs from the highly frequent HLA-B*44:02 allele by amino acid exchanges at residues 77, 80, 81, 82 and 83. We aimed to identify the magnitude of these mismatches on the features of HLA-B*44:09 bound peptides since residues 77, 80 and 81 comprise part of the F pocket which determines sequence specificity at the pΩ position of the peptide. Using soluble HLA technology we determined >200 individual (nonduplicate) self-peptides from HLA-B*44:09 and compared their features with that of the published peptide features of HLA-B*44:02. Both alleles illustrate an anchor motif of E at p2. In contrast to the C-terminal peptide binding motif of B*44:02 (W, F, Y or L), B*44:09-derived peptides are restricted predominantly to L or F. The source of peptides for both alleles is identical (LCL 721.221 cells) allowing us to identify 23 shared peptides. The majority of these peptides however contained the restricted B*44:09 anchor motif of F or L at the pΩ position. Molecular modelling based on the B*44:02 structure highlights that the differences of the C-terminal peptide anchor between both alleles can be explained primarily by the B*44:02(81Ala)?>?B*44:09(81Leu) polymorphism which restricts the size of the amino acid that can be accommodated in the F pocket of B*44:09. These results highlight that every amino acid substitution has an impact of certain magnitude on the alleles function and demonstrate how surrounding residues orchestrate peptide specificity.     

Peptide binding to HLA-A2 and HLA-B27 isolated from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy chain/beta 2-microglobulin complexes requires specific peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.     

Identification of five different Patr class I molecules that bind HLA supertype peptides and definition of their peptide binding motifs

We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20-50% range.     

Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.     

Identification of five new HLA-B*3501-restricted epitopes derived from common melanoma-associated antigens, spontaneously recognized by tumor-infiltrating lymphocytes

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.     

Toward a definition of self: proteomic evaluation of the class I peptide repertoire

MHC class I molecules present host- and pathogen-derived peptides for immune surveillance. Much attention is given to the search for viral and tumor nonself peptide epitopes, yet the question remains, "What is self?" Analyses of Edman motifs and of small sets of individual peptides suggest that the class I self repertoire consists of thousands of different peptides. However, there exists no systematic characterization of this self-peptide backdrop, causing the definition of class I-presented self to remain largely hypothetical. To better understand the breadth and nature of self proteins sampled by class I HLA, we sequenced >200 endogenously loaded HLA-B*1801 peptides from a human B cell line. Peptide-source proteins, ranging from actin-related protein 6 to zinc finger protein 147, possessed an assortment of biological and molecular functions. Major categories included binding proteins, catalytic proteins, and proteins involved in cell metabolism, growth, and maintenance. Genetically, peptides encoded by all chromosomes were presented. Statistical comparison of proteins presented by class I vs the human proteome provides empiric evidence that the range of proteins sampled by class I is relatively unbiased, with the exception of RNA-binding proteins that are over-represented in the class I peptide repertoire. These data show that, in this cell line, class I-presented self peptides represent a comprehensive and balanced summary of the proteomic content of the cell. Importantly, virus- and tumor-induced changes in virtually any cellular compartment or to any chromosome can be expected to be presented by class I molecules for immune recognition.     

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B^*5101 binding self-peptides, 27 paptides bound to HLA-B^*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B^*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B^*5102 and B^*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B^*5102) and that of Gly for Trp at residue 167 (B^*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B^*5102 or HLA-B^*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.     

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.