Cdc37-Hsp90 Complexes Are Responsive to Nucleotide-induced Conformational Changes and Binding of Further Cofactors

Abstract

Hsp90 is an ATP-dependent molecular chaperone, which facilitates the activation and stabilization of hundreds of client proteins in cooperation with a defined set of cofactors. Many client proteins are protein kinases, which are activated and stabilized by Hsp90 in cooperation with the kinase-specific co-chaperone Cdc37. Other Hsp90 co-chaperones, like the ATPase activator Aha1, also are implicated in kinase activation, and it is not yet clear how Cdc37 is integrated into Hsp90 co-chaperone complexes. Here, we studied the interaction between Cdc37, Hsp90, and other Hsp90 co-chaperones from the nematode Caenorhabditis elegans. Nematode Cdc37 binds with high affinity to Hsp90 and strongly inhibits the ATPase activity. In contrast to the human Hsp90 system, we observed binding of Cdc37 to open and closed Hsp90 conformations, potentially reflecting two different binding modes. Using a novel ultracentrifugation setup, which allows accurate analysis of multifactorial protein complexes, we show that cooperative and competitive interactions exist between other co-chaperones and Cdc37-Hsp90 complexes in the C. elegans system. We observed strong competitive interactions between Cdc37 and the co-chaperones p23 and Sti1, whereas the binding of the phosphatase Pph5 and the ATPase activator Aha1 to Cdc37-Hsp90 complexes is possible. The ternary Aha1-Cdc37-Hsp90 complex is disrupted by the nucleotide-induced closing reaction at the N terminus of Hsp90. This implies a carefully regulated exchange process of cofactors during the chaperoning of kinase clients by Hsp90.The ATP-dependent molecular chaperone Hsp90 2 is a ubiquitous protein that interacts with a large number of client proteins, conferring activity or stability to them (1-4). The process of client protein activation of this chaperone requires the hydrolysis of ATP (5, 6). The hydrolysis energy is assumed to be transmitted to the client proteins via conformational changes within Hsp90 (7, 8). Hsp90 contains an N-terminal nucleotide-binding domain (NBD), 3 a middle domain, and a C-terminal dimerization domain. A cyclical reaction pathway, in which Hsp90 goes from an open to a closed conformation, has been proposed for ATP hydrolysis. During this reaction cycle, the two N-terminal domains approach each other and thereby activate the hydrolysis reaction (9, 10). The interaction of the N-terminal domains is initiated by a lid segment (ATP-lid), which closes over the nucleotide-binding pocket and subsequently exposes the N-terminal interaction surfaces (11)(12)(13)(14)(15). This model has been derived from extensive studies of yeast and human Hsp90 proteins and is believed to be conserved for all Hsp90s (14,16,17).Co-chaperone regulation is an important feature of Hsp90 function, and a defined set of co-chaperones has been described for eukaryotic Hsp90 proteins (18). Inhibitory effects on the turnover rate of Hsp90 have been documented for the co-chaperones Sba1/p23 (9,19,20), Sti1/Hop (21, 22), and Cdc37 (23-25), whereas Aha1 and its homolog Hch1 are the only known activa...

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

“…5C). The interaction between CeCdc37 and CeHsp90 was dependent on ionic strength as reported before (25). The interaction of the human proteins also was dependent on ionic strength, but generally, the affinity of complex formation was higher.…”

“…We had previously investigated the interaction between C. elegans Hsp90 (CeHsp90) and its endogenous co-chaperone CeCdc37 (25). There, we had observed an interaction of CeCdc37 with the open and closed conformation of Hsp90, which deviates from interaction modes observed for other eukaryotic Hsp90 systems.…”

“…4C). We did not see any indications of weaker complex formation as we had seen in similar experiments using the C. elegans system (25). We aimed at understanding, whether this is the consequence of using the human Hsp90 protein.…”

“…Cloning, Protein Expression, and Purification-The C. elegans homolog proteins of Cdc37 (CDC-37, W08F4.8), Hsp90 (DAF-21, C47E8.5), and all of their truncation mutants, as well as Aha1 (C01G10.8) and PPH-5 (Y39B6A.2) were obtained as described previously (24,25). The same is true for human Hsp90 and its variants (9), Aha1, Cdc37, and its variants and for yeast Hsp90 (26).…”

“…Aha1 is an ATPase activating cofactor known to bind to the N-and M-domain of Hsp90 (30, 34 -36). In the nematode system, it partially can replace Cdc37 during the kinase chaperone cycle (25). To investigate the binding behavior in the human system, we used our common aUC set up and added hAha1 to a complex formed out of hCdc37* and full-length hHsp90.…”