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 specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-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-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.
Linderoth
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NA
Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USA.
Popowicz
A
A
Sastry
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry*; D001665:Binding Sites; D003432:Cross-Linking Reagents; D006680:HLA Antigens / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D006680:HLA Antigens; D008024:Ligands; D010455:Peptides; C052646:sarcoma glycoprotein gp96 rejection antigens