Transient Interaction of Hsp90 with Early Unfolding Intermediates of Citrate Synthase

Abstract

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively "stabilized" CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.

A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90

“…In the present study, mitochondrial CS was chosen as a model substrate in the analysis of chaperone activity because it is inactive and aggregates rapidly upon incubation at 43 mC [32]. HSP90 binds transiently to unfolding intermediates of the thermally unfolding CS.…”

“…For this mechanism, there are some possibilities as follows : (i) ATP induces a conformational change in HSP90 (from an open to a closed form) [32]. Like many other chaperones, HSP90 is a rather hydrophobic protein, and its hydrophobicity further increases after addition of ATP or heat treatment [32].…”

“…The influence of HSP90 and CDDP on the thermal aggregation of mitochondrial citrate synthase (CS ; Boehringer-Mannheim) at 43 mC was monitored as described [32]. To monitor thermal unfolding\aggregation, the CS concentration was 0n075 µM in 40 mM Hepes buffer (pH 7n4) in the presence or absence of bovine serum albumin (15 µM), HSP90 (0n075 or 0n15 µM), and CDDP (0n15, 1n75 and 1n5 µM).…”

“…To analyse the functional properties of HSP90, we studied its action in protein folding and unfolding reactions in itro. As an assay system, the thermal unfolding and aggregation of mitochondrial CS was used, because CS is inactivated and aggregates rapidly upon incubation at 43 mC [32]. As shown in Figure 4, spontaneous aggregation of CS has been caused at 43 mC and the reaction reached a plateau at 60 min.…”

Conformational dynamics of the molecular chaperone Hsp90

“…Unlike other chaperones, Hsp90 appears to act largely at later stages of the folding pathway (McLaughlin et al, 2002). Unlike the substrates of other chaperones, Hsp90 clients have already achieved a partially folded or almost fully folded conformation before interacting with Hsp90 (Jakob et al, 1995;McLaughlin et al, 2002) suggesting that Hsp90 must adapt its conformation to match each client or that different conformations recognize different clienys. Conformational changes within Hsp90 then induce subtle conformational rearrangements within the client protein facilitating the binding of ligands or partner proteins Young et al, 2001;.…”