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J. Biol. Chem., Vol. 283, Issue 30, 21170-21178, July 25, 2008

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Intra- and Intermonomer Interactions Are Required to Synergistically Facilitate ATP Hydrolysis in Hsp90^*

Christian N. Cunningham¹, Kristin A. Krukenberg², and David A. Agard^¶3

From the Howard Hughes Medical Institute, ^¶Department of Biochemistry and Biophysics, Graduate Group in Biophysics and the Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California 94158

Nucleotide-dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures (Protein Data Bank codes 2IOQ, 2CG9, AND 2IOP) of Hsp90 homologs reveal large scale quaternary domain rearrangements upon the addition of nucleotides. Although previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently. Using the crystal structures as a guide, we examined the role of intra- and intermonomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer, probing the context in which particular residues are important for ATP hydrolysis. Although the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the -phosphate of bound ATP. Small angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP (5'-adenylyl-β,-imidodiphosphate), providing a direct linkage between structural changes and functional consequences. Together, these data indicate that both the cis monomer and the trans monomer and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.

Received for publication, January 3, 2008 , and in revised form, April 24, 2008.

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^* This work was supported by grants from the Howard Hughes Medical Institute and a University of California Discovery Grant (bio03-10401/Agard). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures.

This article was selected as a Paper of the Week.

¹ Supported by an Achievement Rewards for College Scientists fellowship and National Institutes of Health Grant T32 GM08284.

² Supported by a National Defense Science and Engineering Graduate Fellowship.

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³ To whom correspondence should be addressed: 600 16th St., MC2240 Rm. S412D, San Francisco, CA 94158. Tel.: 415-476-2521; Fax: 415-476-1902; E-mail: agard{at}msg.ucsf.edu.

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