The Charged Linker Region Is an Important Regulator of Hsp90 Function

Abstract

Hsp90 is an ATP-dependent molecular chaperone which assists the maturation of a large set of target proteins. Members of the highly conserved Hsp90 family are found from bacteria to higher eukaryotes, with homologues in different organelles. The core architecture of Hsp90 is defined by the N-terminal ATP binding domain followed by the middle domain and the C-terminal dimerization domain. A long, highly charged linker between the N-terminal domain and the middle domain is a feature characteristic for Hsp90s of eukaryotic organisms. We set out to clarify the function of this linker by studying the effects of deletions in this region in vivo and in vitro. Here we show that increasing deletions in the charged linker region lead to defects ranging from mild temperature sensitivity to a lethal phenotype. The lethal deletion variants investigated in this study still exhibit ATPase activity. Deletion of the charged linker ultimately causes a loss of Hsp90 regulation by co-chaperones, as the sensitivity for Aha1-mediated ATPase acceleration declines, and binding of p23/Sba1 is lost in non-viable deletion constructs. In vivo client assays additionally demonstrated that the deletion of the linker had a pronounced effect on the ability of Hsp90 to facilitate client activation. A partial reconstruction of the linker sequence showed that the supplementation by artificial sequences can rescue the functionality of Hsp90 and restore the conformational flexibility of the protein, required for the processing of client proteins. Hsp903 is an ATP-dependent molecular chaperone present in the cytosol of eubacteria and eucaryotes. It regulates the maturation and activation of numerous proteins involved in signal transduction, cell cycle control, hormone signaling, and transcription (1-3). Recent crystal structures of HtpG from Escherichia coli and Hsp90 from Saccharomyces cerevisiae show that the overall structural organization of the proteins is highly conserved (4 -6). Both prokaryotic and eukaryotic Hsp90 proteins consist of an N-terminal ATP binding domain, a middle domain involved in client protein binding, and a C-terminal dimerization domain. During the ATPase cycle, large conformational changes in the Hsp90 dimer lead to the transient dimerization of the N-terminal domains and their association with the middle domains (7-12). Despite the conservation of the basic molecular architecture and the ATPase mechanism, there are major differences between Hsp90 from prokaryotes and eukaryotes. The most striking difference is the emergence of a large set of co-chaperones in eucaryotes that bind to Hsp90 and seem to modulate and expand its properties (13). Furthermore, in contrast to procaryotes, Hsp90 is an essential protein in eucaryotes (14, 15) and it had been shown in S. cerevisiae that ATP hydrolysis by Hsp90 is required for sustaining its essential function (16 -18). Finally, eukaryotic Hsp90 contains additional structural elements compared with prokaryotic Hsp90; that is, a long charged linker between the N terminus and the middle...

The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function

“…Because the CL has been shown to play a crucial role for the function of Hsp90 in vivo (11,15), we set out to analyze the CL substitution mutants in Saccharomyces cerevisiae. Additionally, the variants were tested in a Δcpr7 strain, because Cpr7 becomes essential when the CL is deleted (13).…”

“…The crystal structure of yeast Hsp90 was obtained by partly deleting the CL region and shows a closed, compact conformation in the presence of AMP-PNP [Adenosine 5′-(β,γ-imido)triphosphate] and Sba1/p23 (10). The fact that the CL region is difficult to map structurally led to the assumption that this region is disordered and flexible, thereby enabling the conformational rearrangements of Hsp90 (11,12). Besides the structural indetermination of the CL, its ultimate function remains elusive as well (13).…”

“…Besides the structural indetermination of the CL, its ultimate function remains elusive as well (13). Early studies show that parts of the CL region (amino acids 211-259) are dispensable in yeast (14), whereas more extended deletions affect cell viability, substrate maturation, and regulation by cochaperones (11). These deficiencies can be partially rescued by a short linker consisting of an artificial sequence (11), but its specific amino acid sequence is associated with a gain of function, probably an additional Hsp90 regulatory mechanism (15).…”

Chaperoning parasitism: the importance of molecular chaperones in pathogen virulence

“…Connecting the N and M domains are a number of charged amino acids. This unstructured region, that is highly divergent in both length and sequence among Hsp90 proteins of different species (in contrast to the rest of the protein, which contains large regions that are highly homologous between species), is referred to as the 'charged linker' and plays an essential, although as yet not completely understood role in Hsp90 chaperone function (Hainzl et al 2009;Tsutsumi et al 2009Tsutsumi et al , 2012.…”

A naturally occurring variant of Hsp90 that is associated with decanalization

“…Previous work suggests that this charged region may be important for appropriate peptide binding to the N-terminal of the Hsp90 protein (Scheibel et al 1999) and loss of function of Hsp90 through altered regulation by co-chaperones, although partial reconstruction of this region can help rescue function (Hainzl et al 2009). Presumably a small deletion of a single amino acid in the charged region is likely to have quite subtle effects on Hsp90 function.…”