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

Wanping Xu,Kristin Beebe,Jane B Trepel,Christosomos Prodromou,Matthias P Mayer,Ioannis Gelis,Abbey D Zuehlke,Yinying Lu,Marta Boysen,Dimitra Keramisanou,Juan D Chavez,Len Neckers,James E Bruce

doi:10.1038/s41467-019-10463-y

Abstract

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization 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.

Highlights

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence
Like the Hsp[90] immunoprecipitated with AC88, the Hsp[90] fraction isolated by H90-10 immunoprecipitation was phosphorylated on Y313 to a substantially lesser degree than that seen for Hsp[90] coprecipitated with Aha[1] (Fig. 1a), confirming that Hsp[90] in complex with Aha[1] is preferentially phosphorylated on Y313 compared to the general cellular pool of the chaperone
While increasing Aha[1] association, Y313E phosphomimetic substitution decreased Hsp[90] association with another cochaperone, p60Hop (Fig. 1b). This finding was confirmed by reciprocal immunoprecipitation, in which FLAGp60Hop and HA-Hsp90α were co-transfected into HEK293 cells

Summary

Introduction

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The molecular chaperone heat shock protein 90 (Hsp90) is essential for cellular proteostasis in eukaryotes It facilitates the maturation, stability, and activity of >200 Hsp90dependent proteins, termed clients. Stability, and activity of >200 Hsp90dependent proteins, termed clients The chaperone performs these functions by collaborating with an array of cellular proteins referred to as cochaperones[1]. Hsp[90] is composed of an ATP-binding N-terminal domain (Hsp90-N) that attains ATPase competence upon structural rearrangement of the protein, a middle domain (Hsp90-M) involved in client interaction, and a C-terminal domain (Hsp90C) mediating homodimerization. Binding of Aha[1] facilitates conformational changes in Hsp[90] necessary to establish transient N-domain dimerization, including rearrangement and re-orientation of the catalytic loop in Hsp90-M, repositioning of Hsp[90] N-domain and M-domain, and facilitation of ATP lid closure in Hsp90-N, leading to marked stimulation of Hsp[90] ATPase activity[6,8]

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Conclusion