Abstract
GRP94 is a molecular chaperone that carries immunologically relevant peptides from cell to cell, transferring them to major histocompatibility proteins for presentation to T cells. Here we examine the binding of several peptides to recombinant GRP94 and study the regulation and site of peptide binding. We show that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids. A number of peptides bind to this site with low on- and off-rates and with specificity that is distinct from that of another endoplasmic reticulum chaperone, BiP/GRP78. Binding to the N-terminal fragment is sufficient to account for the peptide binding activity of the entire molecule. Peptide binding is inhibited by radicicol, a known inhibitor of the chaperone activities of HSP90-family proteins. However, the peptide-binding site is distinct from the radicicol-binding pocket, because both can bind to the N-terminal fragment simultaneously. Furthermore, peptide binding does not cause the same conformational change as does binding of radicicol. When the latter binds to the N-terminal domain, it induces a conformational change in the downstream, acidic domain of GRP94, as measured by altered gel mobility and loss of an antibody epitope. These results relate the peptide-binding activity of GRP94 to its other function as a chaperone.
Highlights
GRP94 has long been inferred to be a peptide-binding protein because of its ability to augment presentation of peptides to T cells [1]
We show that peptide binding to this site is inhibited by known ligands of GRP94 and that the inhibition is due to transmission of a conformational change along the chaperone
The characterization of the mode of peptide binding by GRP94 is important because of the ability of this chaperone to mediate presentation of peptides to T cells [31]
Summary
Purified Proteins—Full-length murine GRP94 was expressed and purified as recombinant protein in insect cells. The ⌬355 construct contained amino acids 356 – 802 of murine GRP94 It was expressed in SF9 cells as a His-tagged fusion protein, as described above. In some experiments, binding was measured by direct ␥ counting after separation of the bound and free peptide over spin columns containing P30 beads in buffer A For these experiments, 20 g of protein was used per reaction. Translation products were immunoprecipitated with M1 or M2 monoclonal anti-FLAG (Sigma Chemicals) followed by protein ASepharose (Repligen Corporation, Needham, MA), or 9G10 monoclonal anti-GRP94 (StressGen, Vancouver, BC) followed by protein G-Sepharose (Sigma Chemicals or Pierce) as described in Ref. 30 They were resolved by SDS-PAGE, and the gels were dried, exposed to phosphorimager screens, and recorded using the STORM 820
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