Radicicol-sensitive Peptide Binding to the N-terminal Portion of GRP94

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.

Protein Folding in the Endoplasmic Reticulum

“…As with BiP, the NTD is the adenine nucleotide-binding domain and the nucleotide-binding influences the opening and closing of the chaperone. Geldanamycin, radicicol, and their derivatives bind to the NTD and inhibit the activity of the chaperone by converting the chaperone to its closed conformation (Wearsch et al 1998;Schulte et al 1999;Vogen et al 2002;Soldano et al 2003). The NTD also contains a charged linker domain that supports calcium and cochaperone binding, and controls ATP hydrolysis (Schulte et al 1999;Vogen et al 2002;Hainzl et al 2009).…”

“…Geldanamycin, radicicol, and their derivatives bind to the NTD and inhibit the activity of the chaperone by converting the chaperone to its closed conformation (Wearsch et al 1998;Schulte et al 1999;Vogen et al 2002;Soldano et al 2003). The NTD also contains a charged linker domain that supports calcium and cochaperone binding, and controls ATP hydrolysis (Schulte et al 1999;Vogen et al 2002;Hainzl et al 2009). The MD possesses a large loop that interacts with and controls the ATP-binding site along with a hydrophobic patch important for domain interactions (Dutta and Inouye 2000).…”

An essential role for ATP binding and hydrolysis in the chaperone activity of GRP94 in cells

“…For example, there is no obvious conformational change in the ␤ sheet when apo-GRP94 and nucleotide-bound GRP94 are compared (16,25,39). Yet, indirect biophysical evidence suggests that the ␤ sheet binding site is regulated by both the nucleotide-binding site in the N-terminal domain and by the charged domain that links the N and the M domains (3,7). Possibly, the peptide-binding site is used in vivo for binding a regulatory protein, which is yet to be identified.…”

“…We showed previously that the portion of GRP94 relevant for this activity is the N-terminal and charged linker domains that comprise a peptide-binding domain (5). The ␤ sheet of the N-terminal domain is a peptide-binding site and His-125, within this sheet, is essential for peptide binding in vitro (3,4). His-125 contacts the bound peptide and substitution with aspartate abolished peptide binding (4).…”

“…GRP94 contains four domains: the N-terminal (N) domain contains a nucleotide binding site (1,2), a peptide binding site (3,4), and unmapped sites responsible for interaction with dendritic cells during antigen presentation (5). The second domain, as in HSP90, is a charged linker between the N and middle (M) domains, but it is longer in all GRP94s and functionally important for binding of both nucleotide and calcium (6,7).…”