Aha-type co-chaperones: the alpha or the omega of the Hsp90 ATPase cycle?

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that plays an essential role in cellular homeostasis. It functions in the context of a structurally dynamic ATP-dependent cycle to promote conformational changes in its clientele to aid stability, maturation, and activation. The client activation cycle is tightly regulated by a cohort of co-chaperone proteins that display specific binding preferences for certain conformations of Hsp90, guiding Hsp90 through its functional ATPase cycle. Aha-type co-chaperones are well-known to robustly stimulate the ATPase activity of Hsp90 but other roles in regulating the functional cycle are being revealed. In this review, we summarize the work done on the Aha-type co-chaperones since the 1990s and highlight recent discoveries with respect to the complexity of Hsp90 cycle regulation.

The Hsp90 Chaperone: 1H and 19F Dynamic Nuclear Magnetic Resonance Spectroscopy Reveals a Perfect Enzyme.

Hsp90 is a crucial chaperone whose ATPase activity is fundamental for stabilizing and activating a diverse array of client proteins. Binding and hydrolysis of ATP by dimeric Hsp90 drive a conformational cycle characterized by fluctuations between a compact, N- and C-terminally dimerized catalytically competent closed state and a less compact open state that is largely C-terminally dimerized. We used 19F and 1H dynamic nuclear magnetic resonance (NMR) spectroscopy to study the opening and closing kinetics of Hsp90 and to determine the kcat for ATP hydrolysis. We derived a set of coupled ordinary differential equations describing the rate laws for the Hsp90 kinetic cycle and used these to analyze the NMR data. We found that the kinetics of closing and opening for the chaperone are slow and that the lower limit for kcat of ATP hydrolysis is ∼1 s-1. Our results show that the chemical step is optimized and that Hsp90 is indeed a "perfect" enzyme.

A novel method for site-specific chemical SUMOylation: SUMOylation of Hsp90 modulates co-chaperone binding in vitro.

SUMO is covalently attached to lysine side chains in target proteins by the action of a cascade of E1, E2, and E3 ligases. Unlike ubiquitin, SUMO does not target proteins for degradation but rather plays a regulatory role in activating target proteins or directing them to multiprotein complexes. Isolating SUMOylated proteins from native sources is challenging because of the low stoichiometry of SUMOylation that occurs for any given target protein in cells. Here we report a novel strategy to couple SUMO to the site of a target lysine for the purpose of in vitro study. Introduction of a single cysteine after the C terminal diglycine motif and a cysteine in place of a target lysine in a substrate protein allows for efficient and specific crosslinking of SUMO using a homo-bifunctional maleimide crosslinker. We demonstrate that SUMO can be crosslinked in this manner to amino acid position 178 in the dimeric molecular chaperone, Hsp90. Chemically SUMOylated Hsp90 has very similar ATPase activity compared to unmodified Hsp90 but displays preferential co-chaperone binding in vivo. Our novel strategy can easily be applied to other SUMOylated or ubiquitinated target protein in vitro.

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

Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.

The Mechanism of Hsp90 ATPase Stimulation by Aha1.

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.

A mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p, but not Hch1p.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways.

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.