Molecular mechanisms of peptide loading by the tumor rejection antigen heat shock chaperone gp96 (GRP94)

被引:41
作者
Sastry, S [1 ]
Linderoth, N [1 ]
机构
[1] Rockefeller Univ, Mol Genet Lab, New York, NY 10021 USA
关键词
D O I
10.1074/jbc.274.17.12023
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Complexes of gp96/GRP94 and peptides have been shows to elicit immunogenicity. We used fluorescence to understand peptide association with gp96, A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (similar to 100-150 nM) and forms pyrene excimers, suggesting that the peptides were assembled as dimers. In high salt (2.2 at NaCl), although peptide binding was stronger (K-a approximate to 55 nM) than in low salt, pyrene excimers were absent, implying that peptides were farther apart in the complex. Heat shock-activated peptide binding exhibited characteristics of both low salt and high salt modes of binding. Anisotropy and average lifetime of the bound pyrene suggested that peptides were probably located in a solvent-accessible hydrophobic binding pocket in low salt, whereas in high salt, the peptide may be buried in a less hydrophobic (more hydrophilic) environment. These results suggested that peptide-gp96 complexes were assembled in several different ways, depending on the assembly conditions. Resonance energy transfer between the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or more tryptophan residues were within the critical Forster distance of 27-30 Angstrom from the pyrene in the bound peptide. It is proposed that peptides are assembled within higher order gp96 complexes (dimers, etc.) in a hydrophobic pocket and that there may be a conformational change in gp96 leading to an open configuration for peptide loading.
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页码:12023 / 12035
页数:13
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