The Importance of ATP Binding and Hydrolysis by Hsp90 in Formation and Function of Protein Heterocomplexes

James P Grenert,Brian D Johnson,David O Toft

doi:10.1074/jbc.274.25.17525

Abstract

The chaperone hsp90 is capable of binding and hydrolyzing ATP. Using information on a related ATPase, DNA gyrase B, we selected three conserved residues in hsp90's ATP-binding domain for mutation. Two of these mutations eliminate nucleotide binding, while the third retains nucleotide binding but is apparently deficient in ATP hydrolysis. We first analyzed how these mutations affect hsp90's binding to the co-chaperones p23 and Hop, and to the hydrophobic resin, phenyl-Sepharose. These experiments showed that ATP's effects, specifically, increased affinity for p23 and decreased affinity for Hop and phenyl-Sepharose, are brought on by ATP binding alone. We also tested the ability of hsp90 mutants to assist hsp70, hsp40, and Hop in the refolding of denatured firefly luciferase. While hsp90 is capable of participating in this process in a nucleotide-independent manner, the ability to hydrolyze ATP markedly potentiates hsp90's effect. Finally, we assembled progesterone receptor heterocomplexes with hsp70, hsp40, Hop, p23, and wild type or mutant hsp90. While neither ATP binding nor hydrolysis was necessary to bind hsp90 to the receptor, mature complexes containing p23 and capable of hormone binding were only obtained with wild type hsp90.

Highlights

The 90-kDa heat shock protein1 is an abundant and highly conserved protein involved in a diverse array of cellular processes
Geldanamycin is a specific inhibitor of hsp90, known to disrupt a number of hsp90-dependent processes, including activation of the oncogenic tyrosine kinase pp60vsrc [27], steroid receptor hormone binding [28, 29] and translocation [30], regulation of the HSF1 heat shock transcription factor [31, 32], Cdc37-mediated stabilization of the cyclindependent kinase Cdk4 [33], refolding of denatured firefly luciferase in reticulocyte lysate [12, 34], and ribonucleoprotein complex formation between hepatitis B virus reverse transcriptase and its RNA primer [35]
Two recent studies have shown that mutant hsp90 proteins deficient in either nucleotide binding or hydrolysis are incapable of supporting growth in yeast [36, 37]

Summary

Introduction

The 90-kDa heat shock protein (hsp90)1 is an abundant and highly conserved protein involved in a diverse array of cellular processes. PR complexes were formed by incubation of PR pellets with 20 ␮g of hsp70, 20 ␮g of hsp90, 5 ␮g of Hop, 2 ␮g of ydj-1, and 5 ␮g of p23 in a final volume of 200 ␮l of IB containing 5 mM ATP and 0.01% Nonidet P-40 for 30 min at 30 °C with frequent mixing.

Results

Conclusion