Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex

The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.     

TCR alpha genes direct MHC restriction in the potent human T cell response to a class I-bound viral epitope

The underlying generic properties of alphabeta TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR beta-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ beta-chain was associated with distinct TCR alpha-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR alpha-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR alpha-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.     

Assembly and intracellular trafficking of HLA-B*3501 and HLA-B*3503

Residue 116 of major histocompatibility complex (MHC) class I heavy chains is an important determinant of assembly, that can influence rates of ER-Golgi trafficking, binding to the transporter associated with antigen processing (TAP), tapasin dependence of assembly, and the efficiency and specificity of peptide binding. Here, we investigated assembly and peptide-binding differences between HLA-B*3501(S116) and HLA-B*3503(F116), two alleles differing only at position 116 of the MHC class I heavy chain, that are associated respectively with normal or rapid AIDS progression. A reduced intracellular maturation rate was observed for HLA-B*3503 in HIV-infected and uninfected cells, which correlated with enhanced binding of HLA-B*3503 to TAP. No significant differences in the intrinsic efficiency of in vitro peptide binding by HLA-B*3501 and HLA-B*3503 were measurable with several common peptides or peptide libraries, and both allotypes were relatively tapasin-independent for their assembly. However, thermostability differences between the two allotypes were measurable in a CD4⁺ T cell line. These findings suggest that compared to HLA-B*3501, a reduced intracellular peptide repertoire for HLA-B*3503 could contribute to its slower intracellular trafficking and stronger association with rapid AIDS progression.     

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.     

A human CTL recognizes a caspase-8-derived peptide on autologous HLA-B*3503 molecules and two unrelated peptides on allogeneic HLA-B*3501 molecules

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.     

Structure-based design of nonnatural ligands for the HLA-B27 protein

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and beta-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.     

Peptidic termini play a significant role in TCR recognition

TCR recognition of class I MHC is dependent on the composition of the antigenic peptide and the MHC. Single amino acid substitutions in either the MHC or the peptide may dramatically alter recognition. While the major interactions between TCR and the peptide/MHC complex appear to be focused on the complementarity-determining region (CDR)3, it is also clear from the cocrystal structure of class I MHC and TCR that the amino and carboxyl ends of the peptide may play a role through interactions with the CDR1. In this work we show that gp33 variants substituted at the peptidic termini at the putative CDR1 contact regions show improved recognition in B6 mice. The rank order of recognition is different using the P14 transgenic T cells, suggesting that one reason for improved recognition is a change in the TCR repertoire that recognizes the peptide. However, the affinity of the TCR by some of the peptide/MHC complex with increased recognition is improved, as shown by increased tetramer binding to P14 T cells. These substitutions at the termini of the peptide-binding cleft cause localized conformational changes as seen by changes in mAb binding and crystallographic structures. The different peptide structures also show different conformations in the center of the peptide, but these are shown to be energetically similar and thus most likely have no significance with respect to TCR recognition. Therefore, small conformational changes, localized to the CDR1 contact regions, may play a significant role in TCR recognition.     

Structure-Based Design of Nonnatural Ligands for the HLA-B27 Protein

Abstract

X-ray studies as well as structure-activity relationships indicate that the central part of class I MHC-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, several nonpeptidic spacer have been designed from the X-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the HLA-B*2705 protein was studied by an in vitro stabilisation assay and the thermal stability of all complexes examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove, and thus decrease more or less the affinity of the altered epitopes to HLA-B*2705. Bifunctional spacers ((R)-3-hydroxybutyrate and β-homoalanine combinations) not only bridges the two distant anchoring amino acids but also strongly interact with the binding cleft and lead to an increase in binding to the MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.     

High resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class I. Implications for T-cell receptor engagement and T-cell immunodominance

Although HLA class I alleles can bind epitopes up to 14 amino acids in length, little is known about the immunogenicity or the responding T-cell repertoire against such determinants. Here, we describe an HLA-B*3508-restricted cytotoxic T lymphocyte response to a 13-mer viral epitope (LPEPLPQGQLTAY). The rigid, centrally bulged epitope generated a biased T-cell response. Only the N-terminal face of the peptide bulge was critical for recognition by the dominant clonotype SB27. The SB27 public T-cell receptor (TcR) associated slowly onto the complex between the bulged peptide and the major histocompatibility complex, suggesting significant remodeling upon engagement. The broad antigen-binding cleft of HLA-B*3508 represents a critical feature for engagement of the public TcR, as the narrower binding cleft of HLA-B*3501(LPEPLPQGQLTAY), which differs from HLA-B*3508 by a single amino acid polymorphism (Arg156 --> Leu), interacted poorly with the dominant TcR. Biased TcR usage in this cytotoxic T lymphocyte response appears to reflect a dominant role of the prominent peptide x major histocompatibility complex class I surface.     

Conformational dimorphism of self-peptides and molecular mimicry in a disease-associated HLA-B27 subtype

An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.     

Comparative molecular dynamics analysis of tapasin-dependent and -independent MHC class I alleles

MHC class I molecules load antigenic peptides in the endoplasmic reticulum and present them at the cell surface. Efficiency of peptide loading depends on the class I allele and can involve interaction with tapasin and other proteins of the loading complex. Allele HLA-B*4402 (Asp at position 116) depends on tapasin for efficient peptide loading, whereas HLA-B*4405 (identical to B*4402 except for Tyr116) can efficiently load peptides in the absence of tapasin. Both alleles adopt very similar structures in the presence of the same peptide. Comparative unrestrained molecular dynamics simulations on the alpha(1)/alpha(2) peptide binding domains performed in the presence of bound peptides resulted in structures in close agreement with experiments for both alleles. In the absence of peptides, allele-specific conformational changes occurred in the first segment of the alpha(2)-helix that flanks the peptide C-terminal binding region (F-pocket) and contacts residue 116. This segment is also close to the proposed tapasin contact region. For B*4402, a shift toward an altered F-pocket structure deviating significantly from the bound form was observed. Subsequent free energy simulations on induced F-pocket opening in B*4402 confirmed a conformation that deviated significantly from the bound structure. For B*4405, a free energy minimum close to the bound structure was found. The simulations suggest that B*4405 has a greater tendency to adopt a peptide receptive conformation in the absence of peptide, allowing tapasin-independent peptide loading. A possible role of tapasin could be the stabilization of a peptide-receptive class I conformation for HLA-B*4402 and other tapasin-dependent alleles.     

Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs

The majority of >2000 HLA class I molecules can be clustered according to overlapping peptide binding specificities or motifs recognized by CD8(+) T cells. HLA class I motifs are classified based on the specificity of residues located in the P2 and the C-terminal positions of the peptide. However, it has been suggested that other positions might be relevant for peptide binding to HLA class I molecules and therefore be used for further characterization of HLA class I motifs. In this study we performed large-scale sequencing of endogenous peptides eluted from K562 cells (HLA class I null) made to express a single HLA molecule from HLA-B*3501, -B*3502, -B*3503, -B*3504, -B*3506, or -B*3508. Using sequence data from >1,000 peptides, we characterized novel peptide motifs that include dominant anchor residues extending to all positions in the peptide. The length distribution of HLA-B35-bound peptides included peptides of up to 15 residues. Remarkably, we determined that some peptides longer than 11 residues represented N-terminal-extended peptides containing an appropriate HLA-B35 peptide motif. These results provide evidence for the occurrence of endogenous N-terminal-extended peptide-HLA class I configurations. In addition, these results expand the knowledge about the identity of anchor positions in HLA class I-associated peptides that can be used for characterization of HLA class I motifs.     

Structural modeling of HLA-B*1502/peptide/carbamazepine/T-cell receptor complex architecture: implication for the molecular mechanism of carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis

Drug-induced adverse reactions are a significant problem in healthcare worldwide and are estimated to cost billions of dollars annually in the United States. A portion of such reactions is observed to strongly associate with certain human leukocyte antigen (HLA) alleles; one of the strongest associations is the HLA-B*1502 protein with carbamazepine (CBZ)-induced Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) – the odds ratio value can even be higher than one thousand. The particularly strong association in CBZ-induced SJS/TEN suggests that the HLA-B*1502 is not only a genetic marker but also a participant in the pathogenesis of the disease. In the current study, we attempt to computationally model the atomic-level structure of the complete HLA-B*1502/peptide/CBZ/T-cell receptor (TCR) complex architecture based on prior knowledge obtained from epidemiological investigations as well as in vitro and in vivo assays. The model tells a different story about the molecular mechanism of CBZ-induced SJS/TEN from that previously reported for abacavir (ABC)-induced hypersensitivity (HSR); the CBZ molecule is located at the interface between HLA-B*1502/peptide and TCR, directly contacts the P3–P6 residues of antigen peptide, and bound within a pocket region encompassed by two TCR CDR3 fingers. Molecular dynamics simulation and binding energy analysis further reveal that the CBZ shows considerably high affinity to TCR over HLA-B*1502/peptide, which can tightly interact with the former rather than the latter. From the model, two hypotheses are proposed that can well explain most previous observations and are expected to guide next wet-lab experiments. This study could help to promote our understanding of the molecular mechanism and pathological implication underlying CBZ-induced SJS/TEN.     

The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC.

Cell surface complexes of class I MHC molecules and bound peptide antigens serve as specific recognition elements controlling the cytotoxic immune response. The 2.1 A structure of the human class I MHC molecule HLA-B27 provides a detailed composite image of a co-crystallized collection of HLA-B27-bound peptides, indicating that they share a common main-chain structure and length. It also permits direct visualization of the conservation of arginine as an "anchor" side chain at the second peptide position, which is bound in a potentially HLA-B27-specific pocket and may therefore have a role in the association of HLA-B27 with several diseases. Tight peptide binding to class I MHC molecules appears to result from the extensive contacts found at the ends of the cleft between peptide main-chain atoms and conserved MHC side chains, which also involve the peptide in stabilizing the three-dimensional fold of HLA-B27. The concentration of binding interactions at the peptide termini permits extensive sequence (and probably some length) variability in the center of the peptide, where it is exposed for T cell recognition.     

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.     

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.     

The solvent-inaccessible Cys67 residue of HLA-B27 contributes to T cell recognition of HLA-B27/peptide complexes

Crystallographic studies have suggested that the cysteine at position 67 (Cys(67)) in the B pocket of the MHC molecule HLA-B*2705 is of importance for peptide binding, and biophysical studies have documented altered thermodynamic stability of the molecule when Cys(67) was mutated to serine (Ser(67)). In this study, we used HLA-B27.Cys(67) and HLA-B27.Ser(67) tetramers with defined T cell epitopes to determine the contribution of this polymorphic, solvent-inaccessible MHC residue to T cell recognition. We generated these HLA-B27 tetramers using immunodominant viral peptides with high binding affinity to HLA-B27 and cartilage-derived peptides with lower affinity. We demonstrate that the yield of refolding of HLA-B27.Ser(67) molecules was higher than for HLA-B27.Cys(67) molecules and strongly dependent on the affinity of the peptide. T cell recognition did not differ between HLA-B27.Cys(67) and HLA.B27.Ser(67) tetramers for the viral peptides that were investigated. However, an aggrecan peptide-specific T cell line derived from an HLA-B27 transgenic BALB/c mouse bound significantly stronger to the HLA-B27.Cys(67) tetramer than to the HLA-B27.Ser(67) tetramer. Modeling studies of the molecular structure suggest the loss of a SH ... pi hydrogen bond with the Cys-->Ser substitution in the HLA-B27 H chain which reduces the stability of the HLA-B27/peptide complex. These results demonstrate that a solvent-inaccessible residue in the B pocket of HLA-B27 can affect TCR binding in a peptide-dependent fashion.     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.