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

Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90

30 years of ZMBH

Mit einem Festakt und einem Alumni-Forum begeht das ZMBH am 17. und 18. September 2015 sein 30-jähriges Bestehen. An dieser zentralen Forschungseinrichtung der Ruperto Carola widmen sich Wissenschaftler verschiedener Disziplinen der Erforschung grundlegender molekular- und zellbiologischer Prozesse, die vielfach auch medizinische Aspekte berühren

Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes

The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different KD values. This simultaneous calculation of the KDs from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the KD values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle