Abstract
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.
Highlights
Celiac disease is a complex genetic disorder of the small intestine caused by an inflammatory response to dietary wheat gluten and related proteins from rye and barley [1]
Crystallographic studies demonstrate that the pattern of hydrogen bonding to main chain atoms is similar between major histocompatibility complex (MHC) alleles, and the involved MHC residues are often conserved across MHC alleles
Binding of Norvaline-substituted Peptides—To probe for the potential for hydrogen bonding at positions occupied by proline residues in the ␣I-gliadin epitope, a series of peptides containing norvaline in place of proline was tested for binding to DQ2 in a competitive binding assay
Summary
Peptide Synthesis—N-Methylated peptides used in this study were synthesized using Boc/HBTU chemistry starting from N-␣-t-Boc-L-aminoacyl-phenylacetamidomethyl resin. The peptides were purified by reverse-phase high pressure liquid chromatography on a semi-preparative C18 column using a water-acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid. The labeled indicator peptide (30,000 cpm; 1–5 nM) and various concentrations of unlabeled peptides were incubated with 100 – 300 nM DQ2 overnight at 37 °C in the presence of a mixture of protease inhibitors [25]. Complexes of peptide and DQ2 were separated from unbound peptides by spin column chromatography technique as described previously [27]. Dissociation Experiments—Preformed complexes were prepared by incubating radiolabeled peptide with purified HLA-DQ2 overnight at 37 °C. The complexes were isolated by spin columns and incubated at pH 5.2 in the presence of protease inhibitors (as described in the peptide binding assay) at 37 °C. The dissociation kinetics were fit into the one-phase exponential decay function (Y ϭ As ϫ exp(Ϫksx)) or the two-phase exponential decay function (Y ϭ Af ϫ exp(Ϫkfx) ϩ As ϫ exp(Ϫksx)) using GraphPad Prism (Version 3.02)
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