Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries Ele ́ onore Lepvrier, † Laura Moullintraffort, †,# Michaë l Nigen, ‡ Renan Goude, § Diane Allegro, ∥ Pascale Barbier, ∥ Vincent Peyrot, ∥ Daniel Thomas, † Alexis Nazabal, ⊥ and Cyrille Garnier* ,† † Translation and Folding, UMR-CNRS 6290, Universite ́ de Rennes 1, Campus Beaulieu, 263 Avenue du Gé ne ́ ral Leclerc, 35042 Rennes, Cedex, France ‡ UMR1208 Ingé nierie des Agropolyme ̀ res et Technologies Emergentes INRA-Montpellier SupAgro-CIRAD, Universite ́ Montpellier, 2 Place Pierre Viala, F-34060 Montpellier, France § Microbiologie risques infectieux, EA 1254, Universite ́ de Rennes 1, Campus Beaulieu, 35042 Rennes, Cedex, France ∥ Aix-Marseille Universite ́ , INSERM UMR 911, Centre de Recherche en Oncologie biologique et Onco-pharmacologie, 13385 Marseille, Cedex 5, France ⊥ CovalX AG, 8952 Zü rich-Schlieren, Switzerland * S Supporting Information ABSTRACT: The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultra- centrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time- of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries. T he 90-kDa heat shock protein (Hsp90) is one of the most highly expressed proteins in the cytosol of eukaryotic cells. 1 Hsp90 is highly conserved in evolution and is essential for eukaryotic cell survival. 2 Under stress conditions, Hsp90 is overexpressed. Two isoforms have been identified in the cytosol of eukaryotic cells: the stress-induced α isoform and the constitutively expressed β isoform, 3 with each self-associating as homodimers. Under physiological conditions, Hsp90 is involved in the maturation and activation of over 200 client proteins, operating at later stages of the folding pathway and ensuring that client proteins reach their functional conforma- tion. Some of these client proteins are involved in severe diseases such as cancers and conformation-associated pathol- ogies. 4,5 Under stress conditions, Hsp90 is involved in cell stress tolerance, acting as a holdase for proteins and preventing their irreversible aggregation. 6,7 All of these activities make Hsp90 a major drug target in various diseases. Hsp90 is composed of three domains: the N-terminal domain (NTD), which contains the binding site common to ATP and geldanamycin; 8,9 the middle domain (MD), which is involved in the ATP γ-phosphate hydrolysis and in the binding of client proteins; 10 and the C-terminal dimerization domain (CTD), 11 which contains a binding site for novobiocin and nucleotides. 12 These three domains are connected by highly charged loops, conferring an important intrinsic flexibility to the dimer. Indeed, in solution, apo-Hsp90 is in a conforma- tional equilibrium between open states, the fully open “flying seagull ” and the semiopen “ V shapes ” , 13 and closed conformations through a transient dimerization of the NTDs. 14 Hsp90 dimer structure is modulated by the binding/hydrolysis of nucleotides and by cochaperones. Indeed, numerous cochaperones were found to be progressively grafted onto the theoretical Hsp90 dimer chaperone cycle. 1 Some of these cochaperones are direct activators or inhibitors of Hsp90 ATPase activity, 15 whereas others are involved in the regulation of complexes and in the recruitment of client proteins. 16 Among them is Aha1, the most potent activator of Hsp90 ATPase, which contributes to client protein recruitment. 16−18 Received: December 20, 2014 Accepted: June 13, 2015 Article pubs.acs.org/ac © XXXX American Chemical Society A DOI: 10.1021/acs.analchem.5b00051 Anal. Chem. XXXX, XXX, XXX−XXX