The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes.

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.

The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the gamma-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1-221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.

Differential response in cell proliferation to beta estradiol in coronary arterial vascular smooth muscle cells obtained from mature female versus male animals.

Although the cardioprotective effect of estrogen is well recognized, the mechanisms by which this sex steroid provides a reduction in coronary artery disease are not fully understood. Vascular smooth muscle cells (VSMC) are present in early atherosclerosis and become the dominant cell type. VSMC contain estrogen receptors and may have specific responses to estrogen. We studied the effect of beta-estradiol on the proliferation of coronary VSMC obtained from sexually mature male, female, and oophorectomized pigs. Alpha-estradiol, an inactive isomer of estradiol, had no effect on cells obtained from male or female animals. In vascular smooth muscle cells obtained from sexually mature female animals, significant inhibition of proliferation of coronary vascular smooth muscle cells was noted at physiologic concentrations of beta-estradiol. Progesterone inhibited VSMC proliferation at concentrations of 10(-9)M. In contrast, beta-estradiol did not alter proliferation in porcine coronary vascular smooth muscle cells obtained from sexually mature male or from oophorectomized female animals. This study is the first to indicate, in an animal model, specific gender-related differences in cell proliferation in response to sex steroid hormones.