The Crystal Structure of H-2Db Complexed with a Partial Peptide Epitope Suggests a Major Histocompatibility Complex Class I Assembly Intermediate

Klaus Doering,Tim Elliott,Mayumi Kojima,E. Yvonne Jones,Ann Glithero,Jose Tormo

doi:10.1074/jbc.m511683200

Abstract

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.

Highlights

“anchor residues” make allele specific interactions with polymorphic class I residues located deep inside the binding groove, in “specificitydetermining pockets.”
Class I peptide-binding groove might be between the C terminus of the peptide and the so called F pocket of the binding groove [16, 17]. Using this assay, no binding to H-2Db could be detected for peptides that are shorter than the optimal nonamer by one amino acid residue or more at the N terminus, at concentrations up to 1 mM [8]
We assessed the ability of the C-terminal portion of the Sendai virus nucleoprotein peptide (5NYPAL9) containing both H-2Db anchor residues to support the assembly of soluble H-2Db heavy chain (HC) and ␤2-m in in vitro refolding reactions

Summary

EXPERIMENTAL PROCEDURES

Peptides—FAPGNYPAL corresponds to residues 324 –332 of the Sendai virus nucleoprotein, binds to both H-2Db and H-2Kb, and was synthesized by M. Refolded material was concentrated, purified by gel filtration, and confirmed to be correctly folded H-2Db by immunoprecipitation with mAb B22.249. These purified class I molecules have been defined as “empty” according to several criteria [5] and do not crystallize.. These purified class I molecules have been defined as “empty” according to several criteria [5] and do not crystallize.6 These molecules were mixed with the desired concentration of peptide (HPLC purified to 99%) prior to use in experiments. Thermal Denaturation Measurements—Purified, peptide-receptive H-2Db molecules expressed in CHO cells were diluted to 270 nM in 2 ml of phosphate-buffered saline containing peptide at the specified concentration and were incubated at 4 °C overnight to allow binding.

Statistics for crystallographic data collection and refinement

Side chain

RESULTS

DISCUSSION