Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1

Complex conformational dynamics are essential for function of the dimeric molecular cha- perone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimer- ization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90

The co-chaperone Hch1 regulates Hsp90 function differently than its homologue Aha1 and confers sensitivity to yeast to the Hsp90 inhibitor NVP-AUY922.

Hsp90 is a dimeric ATPase responsible for the activation or maturation of a specific set of substrate proteins termed 'clients'. This molecular chaperone acts in the context of a structurally dynamic and highly regulated cycle involving ATP, co-chaperone proteins and clients. Co-chaperone proteins regulate conformational transitions that may be impaired in mutant forms of Hsp90. We report here that the in vivo impairment of commonly studied Hsp90 variants harbouring the G313S or A587T mutation are exacerbated by the co-chaperone Hch1p. Deletion of HCH1, but not AHA1, mitigates the temperature sensitive phenotype and high sensitivity to Hsp90 inhibitor drugs observed in Saccharomyces cerevisiae that express either of these two Hsp90 variants. Moreover, the deletion of HCH1 results in high resistance to Hsp90 inhibitors in yeast that express wildtype Hsp90. Conversely, the overexpression of Hch1p greatly increases sensitivity to Hsp90 inhibition in yeast expressing wildtype Hsp90. We conclude that despite the similarity between these two co-chaperones, Hch1p and Aha1p regulate Hsp90 function in distinct ways and likely independent of their roles as ATPase stimulators. We further conclude that Hch1p plays a critical role in regulating Hsp90 inhibitor drug sensitivity in yeast

The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation

Hsp90 is a molecular chaperone that acts together with co-chaperones to ensure folding and activation of many client proteins. Here authors show that a N-terminal motif in Aha-type co-chaperones modulates the apparent affinity of Hsp90 for nucleotide substrates

ATPase stimulation of Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

<p>A. Stimulation of the ATPase activity of wildtype Hsp82p (circles), Hsp82p^G313S (squares), and Hsp82p^A587T (triangles) by increasing concentrations of Aha1p. B. Stimulation of the ATPase activity of wildtype Hsp82p (black bars), and Hsp82p^A587T (grey bars) by Hch1p. ATPase rate shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min). Reactions contained 2 µM Hsp82p and indicated concentration of Aha1p or Hch1p.</p

Co-chaperone competition in Hsp82p ATPase reactions.

<p>A. Stimulation of the ATPase activity of wildtype Hsp82p (2 µM) by increasing concentrations of Hch1p in the presence (squares) or absence (circles) of Sba1p (8 µM). ATPase rate shown as a fold increase over intrinsic rate of Hsp82p alone. B. Inhibition of Aha1p-stimulated ATPase rate by increasing concentrations of Sba1p (triangles) and Sti1p (squares). Dashed line represents intrinsic Hsp82p ATPase rate. Reactions contain 2 µM Hsp82p, 4 µM Aha1p and indicated concentrations of Sba1p or Sti1p. ATPase rate shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min). C. Effect of increasing concentrations of Hch1p on the Aha1p-stimulated ATPase activity of wildtype Hsp82p (2 µM) in the presence (squares) or absence (circles) of Sba1p (8 µM). ATPase rate shown as a fold increase over intrinsic rate of Hsp82p alone. Reactions contained 2 µM Hsp82p and indicated concentration of Aha1p or Hch1p.</p

Overexpression of C-terminally myc-tagged Hch1p impairs growth of yeast expressing Hsp82p^A587T but not yeast expressing wildtype Hsp82p.

<p>A. Cells were grown overnight in YPD supplemented with 200 mg/L G418 and then diluted to 1×10⁸ cells per mL. 10-fold serial dilutions were prepared and 5 µL aliquots were spotted on YPD agar plates supplemented with 200 mg/L G418 and grown for 2 days at 30, 35.5 and 37°C. B. Western analysis of yeast shown in 3A with anti-myc tag, anti-His tag and anti-actin antibodies. Levels of mycHch1p (lanes 2, 4, 6, and 8) in strains expressing Hsp82p (lanes 1–4), or Hsp82p^A587T (lanes 5–8) with (lanes 3,4, 7, 8) or without (lanes 1, 2, 5, 6) <i>HCH1</i> deletion. (Anti-myc antibody 9E10).</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Effect of <i>HCH1</i> or <i>AHA1</i> deletion on yeast growth.

*<p>- ipT22Ia appeared to have two copies of HCH1 (possibly due to chromosomal rearrangement or duplication) and was not tested.</p>**<p>- We were unable to obtain a <i>HCH1</i> deletion for this strain.</p

Inhibition of Aha1p-stimulated ATPase activity of Hsp82p, Hsp82p^G313S and Hsp82p^A587T.

<p>A. Inhibition of Aha1p-stimulated ATPase activity of wildtype Hsp82p (circles), Hsp82p^G313S (squares), and Hsp82p^A587T (triangles) by increasing concentrations of Sba1p. ATPase rate for wildtype Hsp82p and Hsp82p^A587T shown on left axis (Reactions contained 2 µM Hsp82p, 10 µM Aha1p and indicated concentrations of Sba1p). ATPase rate for Hsp82p^G313S shown on right axis (reactions contained 5 µM Hsp82p, 10 µM Aha1p and indicated concentrations of Sba1p). B. Inhibition of Hch1p-stimulated ATPase activity of wildtype Hsp82p (circles), and Hsp82p^A587T (triangles) by increasing concentrations of Sba1p. Reactions contained 2 µM Hsp82p, 10 µM Hch1p and indicated concentrations of Sba1p. C. Inhibition of Aha1p-stimulated ATPase activity of wildtype Hsp82p (black bars) and Hsp82p^A587T (white bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. D. Inhibition of Aha1p-stimulated ATPase activity of Hsp82p^G313S (grey bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. E. Inhibition of Hch1p-stimulated ATPase activity of wildtype Hsp82p (black bars) and Hsp82p^A587T (white bars) by Sti1p. All reactions contained 2 µM Hsp82p and indicated concentrations of co-chaperones. ATPase rates shown in µM ATP hydrolyzed per minute per µM of enzyme (1/min).</p