ATP-binding Properties of Human Hsp90

Abstract

Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion.Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with halfmaximal binding at 400 M ATP was detected. The functional significance of this weak interaction remains enigmatic.Under stress conditions, e.g. high temperatures, cells overexpress a distinct set of proteins, the so-called heat shock or stress proteins. The major classes of heat shock proteins, Hsp90, 1 Hsp70, Hsp60, and small Hsps, are thought to function as molecular chaperones during protein folding (1-3). The mechanisms of these chaperone functions are still under intensive investigation. In the case of Hsp70 and Hsp60, ATP binding and hydrolysis is a major requirement in chaperone-mediated folding (1). In vivo experiments suggest that Hsp90, one of the most abundant and conserved heat shock proteins, is a specific chaperone involved in regulating signal transduction pathways by assisting structural changes of certain kinases and steroid receptors (4 -8). In addition, results from in vitro studies highlight the general chaperone activities of Hsp90 (2, 3). Like other chaperones, Hsp90 performs at least part of its activity in association with specific partner proteins (9 -14), some of which seem to function as molecular chaperones themselves (15,16). In this context, the involvement of ATP in Hsp90 function is still a controversial subject (11)(12)(13)(17)(18)(19), and ATP binding as well as ATPase activity of Hsp90 have been reported previously (18, 20 -23). In contrast to these findings, we have shown that yeast Hsp90 does not bind ATP (17) by means of assays that are reflecting structural changes of the observed protein in the presence of ligand, detecting binding of the protein to immobilized ATP or binding of fluorescencelabeled ATP analogues to Hsp90. These observation enabled only the investigation of tight interactions between ATP and Hsp90. New results concerning p23, a partner protein of Hsp90 that is thought to play an important role in Hsp90/steroid receptor complexes, readdressed the possibility that human Hsp90 is an ATP-binding protein (18). This brought up the question of whether human Hsp90 differs in its ATP...

Comparative analysis of the ATP‐binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C‐terminal ATP‐binding site

“…It has been reported that Hsp90 cannot bind strongly to adenine-modified nucleotide analogs, but interacts with ribose-modified ATP with an affinity comparable to that of unmodified ATP [6]. Therefore we studied the interaction of differently substituted nucleotides with Hsp90.…”

“…On the contrary, it does not show a significant interaction with GTP, pirimidine nucleotides, and nucleotides in which the ribose-2¢-OH position has been substituted (TNP, ribose-attached resin; phosphate in NADP). The integrity of the adenine ring is also important for binding, since Hsp90 does not bind to C8-linked ATPresins under stringent conditions ( [6,13]; Cs. S} o oti and P. Csermely, unpublished observations), and a substitution at the 6-adenine position (e.g.…”

“…On the contrary, recent experiments on N-terminally truncated Hsp90 constructs suggested that GTP, indeed, may bind to the C-terminal domain [19]. Using different fluorescent ATP analogs, including N 6 -etheno-ATP, Scheibel et al [6] could not detect a high affinity ATP-binding to Hsp90. However, they could see a weak binding to an ATP-analog spinlabeled on the ribose hydroxyls [6].…”

“…Using different fluorescent ATP analogs, including N 6 -etheno-ATP, Scheibel et al [6] could not detect a high affinity ATP-binding to Hsp90. However, they could see a weak binding to an ATP-analog spinlabeled on the ribose hydroxyls [6]. Unfortunately, the question, whether GA inhibited this interaction was not addressed.…”

“…Hsp90 is an ATP-binding chaperone [5,6] and ATP binding induces a conformational change in Hsp90 [7,8]. Assembly of the Hsp90-organized chaperone machinery, the foldosome, with target proteins requires ATP [9,10]; moreover, ATP binding and hydrolysis are essential for the in vivo function of Hsp90 [11,12].…”