GRP94, the endoplasmic reticulum Hsp90 paralog, binds a diverse array of
peptides, a subset of which are suitable for assembly onto nascent
MHC class I molecules. At present, the mechanism, site, and regulation of
peptide binding to
GRP94 are unknown. Using VSV8, the immunodominant
peptide epitope of the
vesicular stomatitis virus, and native, purified
GRP94, we have investigated GRP94-peptide complex formation. The formation of stable GRP94-VSV8 complexes was slow; competition studies demonstrated that
peptide binding to
GRP94 was specific. VSV8 binding to
GRP94 was stimulated 2-fold or 4-fold, respectively, following chemical denaturation/renaturation or transient heat shock. The activation of GRP94-peptide binding occurred coincident with a stable, tertiary conformational change, as identified by
tryptophan fluorescence and proteolysis studies. Analysis of
GRP94 secondary structure by circular dichroism spectroscopy indicated an identical alpha-helical content for the native, chemically denatured/renatured, and heat-shocked forms of
GRP94. Through use of the environment-sensitive fluorophores
acrylodan and
Nile Red, it was observed that the activation of
peptide binding was accompanied by enhanced
peptide and
solvent accessibility to a hydrophobic binding site(s).
Peptide binding to native or activated
GRP94 was identical in the presence or absence of
ATP or
ADP. These results are discussed with respect to a model in which
peptide binding to
GRP94 occurs within a hydrophobic binding pocket whose accessibility is conformationally regulated in an
adenine nucleotide-independent manner.