Structure of the N-terminal Domain of GRP94

Abstract

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 Å of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.

Ligand-induced Conformational Shift in the N-terminal Domain of GRP94, an Hsp90 Chaperone

“…enosine (NECA), radicicol, and 2-chlorodideoxyadenosine, all of which were essentially identical to one another except for the different ligands (26). Although refined independently, the ATP⅐GRP94, ADP⅐GRP94, and AMP⅐GRP94 complexes are also nearly identical to one another.…”

“…1B. The adenine moiety of the nucleotide fits into the ligand binding cavity in a manner similar to that of NECA (26) and makes similar contacts with the protein. In contrast to NECA, however, the 5Ј substituent of ADP does not interact with the second binding pocket.…”

“…In the previously determined structure of the closed conformation of GRP94, a second pocket adjacent to the adenine binding cavity was identified as the binding site for the 5Ј substituent of NECA (26). In the open conformation, the repositioning of helix 4 exposes this second ligand binding pocket but at the same time renders it sterically incompatible with NECA binding.…”

“…These differences suggest that the structure of GRP94 may reveal an alternative regulatory mechanism for GRP94. The structure of the N-domain of GRP94 in complex with a series of inhibitory ligands was reported recently and revealed that, unlike Hsp90, the inhibitor-bound protein was sterically incompatible with adenosine nucleotide binding (26). This indicated that ATP binding would lead to a conformational change in the N-domain of GRP94, although the extent and nature of this change was unknown.…”

Dynamics of conserved waters in human Hsp90: implications for drug design

“…Several crystal structures of Hsp90 are available (Prodromou et al 1997a,b;Stebbins et al 1997;Obermann et al 1998;Roe et al 1999;Jez et al 2003;Soldano et al 2003;Wright et al 2004); in all cases the active site contains some bound water molecules. The structural, dynamical and functional importance of water molecules for biomacromolecular structure and recognition is well appreciated.…”