The Mechanism of Hsp90 ATPase Stimulation by Aha1.

Annemarie Wolmarans,Brian Lee,Leo Spyracopoulos,Paul Lapointe

doi:10.1038/srep33179

Abstract

Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer.

Highlights

Hsp[90] is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’
We report here that the E33A mutation blocks the ability of Aha1p to stimulate ATP hydrolysis in the opposite subunit of a heterodimer
Aha1p N terminal domain (Aha1pN) can exert a small stimulatory effect from either subunit of a heterodimer harbouring one Hsp82pE33A subunit. This suggests that the E33A mutation blocks the rearrangement of the Hsp[90] N domains that is required for the action of the Aha1p C domain

Summary

Introduction

Hsp[90] is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’. Client maturation by the Hsp[90] dimer occurs in the context of an ATP-driven functional cycle during which Hsp[90] undergoes global conformational rearrangements that involve inter- and intra-protomer interactions[14,15,16]. The most potent stimulator of the low ATPase activity of Hsp[90] is Aha[1], or the “activator of Hsp[90] ATPase’’19,28–30 This co-chaperone has been shown to play a critical role in kinase activation and membrane protein folding in mammalian cells, the mechanism of Aha[1] action is poorly understood[11,31]. Yeast possess a co-chaperone called Hch1p that is homologous to the Aha1p N terminal domain (Aha1pN) (Fig. 1B), which is useful for interrogating domain rearrangements that occur upon interaction with the middle domain[19,28,29,34]. Comparing Hch1p and Aha1p can provide biological insight into the mechanics of Hsp[90] regulation

Methods

Results

Conclusion