Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).     

Thermodynamics of phosphotyrosine peptide–peptoid hybrids binding to the p56lck SH2 domain

A frequently used approach to transform peptides into more drug‐like compounds is preparation of the corresponding peptoids or peptide–peptoid hybrids. Although peptoids have advantages, there may also be some disadvantages such as their increased flexibility and the reduced ability for hydrogen bond formation due to alkylation of the backbone amide nitrogen, which might affect the free Gibbs energy (ΔG). To obtain more insight into these contributions to ΔG, we performed thermodynamic analyses on the interaction between peptide–peptoid hybrids, based on the sequence ‐pTyr‐Glu‐Glu‐Ile‐, and the p56^lck (Lck) Src homology 2 domain. van't Hoff analysis was performed on binding data obtained from surface plasmon resonance competition experiments in a temperature range of 10–40 °C. It is observed that amino acid–peptoid substitutions do not have a systemic negative effect on the entropic contributions to ΔG. However, loss in hydrogen‐bonding capacity of the backbone may strongly reduce the binding enthalpy and contribute to the observed lower binding affinity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Cutting edge: TCR contacts as anchors: effects on affinity and HLA-DM stability

Peptides presented via the class II MHC (MHCII) pathway are selected based on affinity for MHCII and stability in the presence of HLA-DM. Currently, epitope selection is thought to be controlled by the ability of peptide to sequester "anchor" residues into pockets in the MHCII. Residues exhibiting higher levels of solvent accessibility have been shown to contact TCR, but their roles in affinity and complex stability have not been directly studied. Using the HLA-DR1-binding influenza peptide, hemagglutinin (306-318), as a model, we show that side chain substitutions at these positions influence affinity and HLA-DM stability. Multiple substitutions reduce affinity to a greater extent than the loss of the major P1 anchor residue. We propose that these effects may be mediated through the H-bond network. These results demonstrate the importance of solvent-exposed residues in epitope selection and blur the distinctions between anchor and TCR contact residues.     

The effect of a side chain-backbone swap on protein stability.

To assess the relative importance of backbone hydrogen bonding (H-bonding) vs. side chain hydrophobicity in protein structural formation, a method called side chain-backbone swap is proposed. Such a method swaps the side chain and backbone portions of certain amino acid residues, such as Asp, Glu, Asn, Gln, Lys, and Arg. Such a swap retains the sequence of a polypeptide and preserves the identity of the backbone linkage. On the other hand, the swap disrupts backbone H-bonding geometry because of the introduction of extra methylene groups into the peptide backbone. In this project, we chose the two-stranded alpha-helical coiled-coil to implement side chain-backbone swap. A pair of 36-residue peptides was designed. The two peptides have identical sequence with four residues in each heptad repeat occupied by glutamyl residues. Each glutamic acid was incorporated either as alpha-glutamyl residue (the peptide is denoted as alpha-Glu-36) or as gamma-glutamyl residue (the peptide is denoted as gamma-Glu-36). The inter-conversion between the two peptides constitutes a side chain-backbone swap. Residues constituting the hydrophobic core of the coiled-coil, however, are left unchanged. The peptide pair was characterized by circular dichroism spectroscopy, reversed-phase liquid chromatography (RPLC), and two-dimensional nuclear magnetic resonance (NMR). The results indicate that alpha-Glu-36 is a two-stranded alpha-helical coiled-coil while gamma-Glu-36 lacks stable structural elements. It is concluded that, at least for coiled-coils where hydrophobic interactions are predominantly long-range, local backbone H-bonding is a required for structural formation, consistent with a hierarchic folding mechanism. The methodological implication of side chain-backbone swap is also discussed.     

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.     

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

Simulation Studies on the Stabilities of Aggregates Formed by Fibril-Forming Segments of α-Synuclein

Abstract

We performed molecular dynamics simulations for various oligomers with different β-sheet conformations consisting of α-Synuclein 71–82 residues using an all atom force field and explicit water model. Tetramers of antiparallel β-sheet are shown to be stable, whereas parallel sheets are highly unstable due to the repulsive interactions between bulky and polar side chains as well as the weaker backbone hydrogen bonds. We also investigated the stabilities of double antiparallel β-sheets stacked with asymmetric and symmetric geometries. Our results show that this 12 amino acid residue peptide can form stable β-sheet conformers at 320K and higher temperatures. The backbone hydrogen bonds in β-sheet and the steric packing between hydrophobic side chains between β-sheets are shown to give conformational stabilities.     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the − NH − groups has been replaced by − CH₂−) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

Ovalbumin(323-339) peptide binds to the major histocompatibility complex class II I-A(d) protein using two functionally distinct registers

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.     

Solution structure of BmKK4, the first member of subfamily alpha-KTx 17 of scorpion toxins

BmKK4 is a 30 amino acid peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch. It has been classified as the first member of scorpion toxin subfamily alpha-KTx 17. The 3D structure of BmKK4 in solution has been determined by 2D NMR spectroscopy. This toxin adopts a common alpha/beta-motif, but shows a distinctive local conformation. The most novel feature is that the regular arrangements of the side chains of the residues involved in the beta-sheet of BmKK4 are distorted by a classic beta-bulge structure, which involves two residues (Asp18 and Arg19) in the first strand opposite a single residue (Tyr26) in the second strand. The bulge produces two main changes in the structure of the antiparallel beta-sheet: (1) It disrupts the normal alteration of the side chain direction; the side chain of Asp18 turns over to form a salt bridge with that of Arg19. (2) It accentuates the twist of the sheet, and alters the direction of the antiparallel beta-sheet. The unusual structural feature of the toxin is attributed to the shorter peptide segment (Leu15-Arg19) between the third and fourth Cys residues and two unique residues (Asp18 and Arg19) at the position preceding the fourth Cys. In addition, the lower affinity of the peptide for the Kv channel is correlated to the structural features: residue Arg19 instead of a Lys residue at the critical position for binding and the salt bridge formed between residues Arg19 and Asp18.     

Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding

Major histocompatibility complex (MHC) glycoproteins play an important role in the development of an effective immune response. An important MHC function is the ability to bind and present 'processed antigens' (peptides) to T cells. We show here that the purified human class II MHC molecule, HLA-DR1, binds peptides that have been shown to be immunogenic in vivo. Detergent-solubilized HLA-DR1 and a papain-cleaved form of the protein lacking the transmembrane and intracellular regions have similar peptide binding properties. A total of 39 single substitutions were made throughout an HLA-DR1 restricted hemagglutinin epitope and the results determine one amino acid in this peptide which is crucial to binding. Based on this analysis, a synthetic peptide was designed containing two residues from the original hemagglutinin epitope embedded in a chain of polyalanine. This peptide binds to HLA-DR1, indicating that the majority of peptide side chains are not required for high affinity peptide binding.     

Structural definition of the H-2Kd peptide-binding motif

Classic major histocompatibility complex (MHC) proteins associate with antigen- and self-derived peptides in an allele-specific manner. Herein we present the crystal structure of the MHC class I protein H-2K(d) (K(d)) expressed by BALB/c mice in complex with an antigenic peptide derived from influenza A/PR/8/34 nucleoprotein (Flu, residues 147-155, TYQRTRALV). Analysis of our structure in conjunction with the sequences of naturally processed epitopes provides a comprehensive understanding of the dominant K(d) peptide-binding motif. We find that Flu residues Tyr(P2), Thr(P5), and Val(P9) are sequestered into the B, C, and F pockets of the K(d) groove, respectively. The shape and chemistry of the polymorphic B pocket make it an optimal binding site for the side chain of Tyr(P2) as the dominant anchoring residue of nonameric peptides. The non-polar F pocket limits the amino acid repertoire at P9 to hydrophobic residues such as Ile, Leu, or Val, whereas the C pocket restricts the size of the P5-anchoring side chain. We also show that Flu is accommodated in the complex through an unfavorable kink in the otherwise extended peptide backbone due to the presence of a prominent ridge in the K(d) groove. Surprisingly, this backbone conformation is strikingly similar to D(b)-presented peptides despite the fact that these proteins employ distinct motif-anchoring strategies. The results presented in this study provide a solid foundation for the understanding of K(d)-restricted antigen presentation and recognition events.     

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.     

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.     

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.     

Membrane channel forming polypeptides. 270-MHz proton magnetic resonance studies of the aggregation of the 11-21 fragment of suzukacillin in organic solvents

270-MHz 1H NMR studies of the 11-21 suzukacillin fragment Boc-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (11-G) and its analogue Boc-Ala-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-OMe (11-A) have been carried out in CDCl3 and (CD3)2SO. The NH chemical shifts and their temperature coefficients have been measured as a function of peptide concentration in both solvents. It is established that replacement of Gln by Ala is without effect on backbone conformation. Both peptides adopt highly folded 310 helical conformations stabilized by seven intramolecular 4 leads to hydrogen bonds. Nonlinear temperature dependences are demonstrated for free NH groups in the Gln(1) peptide. Aggregation is mediated by intermolecular hydrogen bonds formed by solvent-exposed NH groups. A major role for the Gln side chain in peptide association is suggested by differences in the NMR behavior of the Gln(1) and Ala(1) peptides. For the Gln(1) peptide in CDCl3, the carboxamide side chain carbonyl group forms an intramolecular hydrogen bond to the peptide backbone, while the trans side chain NH shows evidence for intermolecular interactions. In (CD3)2SO, the cis carboxamide NH is involved in intermolecular hydrogen bonding. The possible role of the central Gln residue in stabilizing aggregates of peptide channel formers is discussed, and a model for hexameric association is postulated.     

Mimetics of a T cell epitope based on poly-N-acylated amine backbone structures induce T cells in vitro and in vivo

Peptidomimetics of the major histocompatibility complex (MHC) class I-restricted ovalbumin-derived T cell epitope SIINFEKL were generated by replacing parts of the peptide backbone by a poly-N-acylated amine (PAA) backbone with aromatic, heteroaromatic, and pseudoaromatic side chains that branch off of the main chain at the amine nitrogen. The structure of the PAAs was designed to position this side chain in the central epitope anchor pocket of the MHC molecule. A number of biologically active PAAs were found that induced cytolysis by the mouse cytotoxic T cell clone 4G3. Competition experiments with independent peptides that are known to bind to the restricting MHC molecule H-2K(b) suggest that the PAAs are bound by the MHC molecules at the same site as conventional peptide epitopes. The PAAs were active also in vivo and induced primary cytotoxic T cell responses in mice.     

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.     

Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the celiac disease-associated HLA-DQ2 molecule

Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.