Reference: Wegele H, et al. (2003) Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90. J Biol Chem 278(41):39303-10

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Abstract


Hsp90 is a dimeric, ATP-regulated molecular chaperone. Its ATPase cycle involves the N-terminal ATP binding domain (amino acids (aa) 1-272) and, in addition, to some extent the middle domain (aa 273-528) and the C-terminal dimerization domain (aa 529-709). To analyze the contribution of the different domains and the oligomeric state on the progression of the ATPase cycle of yeast Hsp90, we created deletion constructs lacking either the C-terminal or both the C-terminal and the middle domain. To test the effect of dimerization on the ATPase activity of the different constructs, we introduced a Cys residue at the C-terminal ends of the constructs, which allowed covalent dimerization. We show that all monomeric constructs tested exhibit reduced ATPase activity and a decreased affinity for ATP in comparison with wild type Hsp90. The covalently linked dimers lacking only the C-terminal domain hydrolyze ATP as efficiently as the wild type protein. Furthermore, this construct is able to trap the ATP molecule similar to the full-length protein. This demonstrates that in the ATPase cycle, the C-terminal domain can be replaced by a cystine bridge. In contrast, the ATPase activity of the artificially linked N-terminal domains remains very low and bound ATP is not trapped. Taken together, we show that both the dimerization of the N-terminal domains and the association of the N-terminal with the middle domain are important for the efficiency of the ATPase cycle. These reactions are synergistic and require Hsp90 to be in the dimeric state.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Wegele H, Muschler P, Bunck M, Reinstein J, Buchner J
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