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

Abstract

The hsp90 1 family of molecular chaperones is required for the activation of a large number of proteins, including those involved in eukaryotic cell cycle regulation, signal transduction, immune response, and transcription (1, 2). GRP94 (also known as gp96), the endoplasmic reticulum paralog of cytoplasmic Hsp90, participates in the maturation of proteins that are transported to the cell surface, including Toll-like receptors and certain integrins (3), or secreted, such as IgGs (4, 5). The activity of the hsp90 family chaperones is regulated by ATP (6). Fungally derived ansamycin antibiotics such as geldanamycin and radicicol (7) compete for the ATP binding site (8 -11) and exhibit potent anti-tumor activity by acting as pan-hsp90 inhibitors (12-17). Given their central importance in the biology of protein folding and in cellular stress response, and their potential as targets in a variety of therapeutic strategies, the mechanism by which hsp90 chaperone activity is regulated and the chemistry of their interactions with client substrates are under intensive investigation.GRP94 and Hsp90 exist as obligate homodimers, with each subunit consisting of an N-terminal regulatory and ligand binding domain, a charged region, a middle domain, and a C-terminal dimerization domain. This organization and the structural similarity of their N-terminal ATP binding domains characterize the GHKL family of proteins (18), whose members in addition to Hsp90s also include DNA gyrase, histidine kinases, DNA topoisomerase II, and MutL. By analogy to DNA gyrase (19) and MutL (20), it has been proposed that Hsp90 chaperone activity is linked to an N-domain conformational change that is coupled to ATP binding and hydrolysis and that results in the dimerization of the N-domains (21, 22). Mechanistic explanations of how ATP binding and hydrolysis are coupled to Hsp90 activity have been hindered, however, by the lack of any observed change in the conformation of the Ndomain in response to ligands. The complete protein, rather than just the isolated N-domain, is required for ATP hydrolysis (6, 23), suggesting that conformational rearrangements resulting in a close interaction between the N-and middle domains are important for Hsp90 activity. Interactions with co-chaperones may also be important catalysts for ATP hydrolysis in human Hsp90 as well (24).GRP94 and Hsp90 are greater than 50% identical in their N-domains, yet the biochemical hallmarks of Hsp90 chaperone activity are largely missing for this paralog. ATP hydrolysis above background levels has not been detected for GRP94 (25), nor have co-chaperones or partner proteins been identified. In addition, although Hsp90 binds ADP/ATP with micromolar affinity (11), the binding constant for adenosine nucleotides to GRP94 is estimated to be of several millimolar units (25). 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...

Understanding ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution

“…Indeed, the subsequent crystal structures of the ATP-bound full-length dimer have revealed a major conformational change, involving swapping of the N-terminal chain and lid refolding on the ATP-binding site. The comparison of our modeled Hsp90 NTD structures with the crystal structures of GRP94, the ER paralog of Hsp90, in the complex with ATP (PDB entry 1TC0) (25), the apo form (PDB entry 2YT2) (37), and the structure of yeast Hsp90 (PDB entry 1AH6) (38) has confirmed that in the isolated N-domain the lid is in the open conformation. The inhibitor bound complexes were obtained starting from the complex of human Hsp90 in complex with geldanamycin (PDB entry 1YET) (16), removing the inhibitor and docking Shepherdin, Shepherdin[79 -83] or AICAR as described in refs.…”

“…It is worth noting that ATP-induced conformational changes have been also observed in GRP94, the endoplasmatic reticulum paralog of Hsp90 (25). In this case, ATP binding was shown to induce dramatic conformational rearrangements in the helices 1, 4, and 5 (corresponding to the N-terminal helix and the ATP-lid), which in turn resulted in the assembly of a second NTD in a functional dimer (25). In contrast, we have found that deletion of the terminal phosphate group in the ADP complex and binding with the inhibitors can minimize repulsive interactions ( Fig.…”

“…Interestingly, the ⌬1-24 deletion results in the complete loss of the ATPase activity, showing that reactivity of the chaperone depends on the intradomain dynamical coupling of the N-terminal and ATP-lid regions of Hsp90 (18). It is worth noting that ATP-induced conformational changes have been also observed in GRP94, the endoplasmatic reticulum paralog of Hsp90 (25). In this case, ATP binding was shown to induce dramatic conformational rearrangements in the helices 1, 4, and 5 (corresponding to the N-terminal helix and the ATP-lid), which in turn resulted in the assembly of a second NTD in a functional dimer (25).…”

Conformational dynamics of the molecular chaperone Hsp90

“…The endoplasmic reticulum homolog, Grp94, has a unique 5 amino acid insertion in the lid (residues 182-186) leading to a structural rearrangement of the lid in response to nucleotide that is different from other Hsp90 homologs ( Figure 4C). Although the lid position is unique in nucleotide-bound Grp94, this conformation also leads to the exposure of a large predominantly non-polar surface (Immormino et al, 2004) as seen in AMPPNPbound yHsp90. The hydrophobic surface may also serve as an interaction site for NTD dimerization in Grp94.…”

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

“…The various crystal structures of GRP94 and Hsp90 do not ascribe a clear function to the N-terminal ␤ sheet. 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).…”