Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94)

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specif ic type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous pept ides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derive d pep tide ligand bearing a photoreactive azido group was specifically boun d by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-bindi ng site was mapped by specific proteolysis of the cross-links followed by a nalysis of the cross-linked peptides using a judicious combination of SDS-g el electrophoresis, mass spectrometry, and amino acid sequencing. The minim al peptide-binding site was mapped to amino acid residues 624-630 in a high ly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major hist ocompatibility complex class I molecule bound to a similar peptide. The gp9 6-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-she ets. Interestingly, in this model, the peptide binding pocket abuts the dim erization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptide s to major histocompatibility complex class I molecule for antigen presenta tion.