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

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.

Fig. 1

Y313 phosphorylation-promoted Hsp90–Aha1 interaction is mediated by Aha1-C. a Hsp90-associated Aha1 is phosphorylated on Y313 to a higher level compared to the general Hsp90 pool. Hsp90 was immunoprecipitated from transfected 293A cells with specific antibodies (H90-10 or AC88) or co-precipitated with FLAG-tagged Aha1. Hsp90 protein levels were examined by western blot, and Y313 phosphorylation was detected with an antibody specific for the phosphorylation of this site. b Y313 phosphomimetic substitution increases Hsp90 interaction with Aha1 but decreases the interaction with p60^Hop. FLAG-tagged Hsp90 was immunoprecipitated from transfected cells. Co-precipitated endogenous Aha1, p23, and p60^Hop were detected by western blot. Precipitated Hsp90 proteins were stained with Coomassie Blue (CBB). c Y313E substitution decreases Hsp90 association with p60^Hop. FLAG-p60^Hop was co-expressed with HA-Hsp90 and was immunoprecipitated from cells. Hsp90 and p60^Hop were detected by western blot with indicated antibodies. d Y313E phosphomimetic substitution in Hsp90 increases association of wild-type Aha1 and Aha1-C but not Aha1-N. FLAG-tagged Aha1 (wild-type or individual domains) were co-expressed with HA-tagged Hsp90, and complexes were immunoprecipitated with anti-FLAG resin. Exogenous Hsp90 was detected by western blot with anti-HA antibody. E = glutamic acid, F = phenylalanine. Bottom: schematic illustration of the Aha1 protein. e Point mutations in Aha1-C substantially decrease association with Hsp90-Y313E. Experiments were performed as in d. Endogenous co-precipitated Hsp90 (wild-type) was discerned with the antibody SPS-771, which significantly favors recognition of untagged Hsp90. Bottom: Schematic illustration of Aha1 with point-mutations indicated. f K273A mutation in Aha1-C abolishes the stimulatory effect of Y313 phosphomimetic substitution on Aha1 association. Experiments were performed as in d. Please see Supplementary Fig. 1 for more information. Source data for this figure are provided as a Source Data File

Fig. 2

Aha1 interaction with Hsp90-Y313E is independent of Hsp90 N-domain closure. a Schematic illustration of Hsp90 monomer and dimer, showing the boundaries of the domains and the location of the mutations used in this figure. b Dimerization-inhibiting mutation in Hsp90 fails to counteract the stimulatory effect of Y313E on Aha1 interaction. FLAG-tagged Hsp90, wild-type, or indicated mutants were expressed in 293A cells, immunoprecipitated with the M2 anti-FLAG antibody and verified with CBB staining after electrophoresis. Co-precipitated Aha1 was examined by western blot. c The H210A mutation in Hsp90-N differentially affects Aha1 interaction with wild-type and Y313E Hsp90. Immunoprecipitation and western blot for indicated proteins were performed as in b. d The H210A mutation in Hsp90 completely abolishes the effect of the closed conformation-inducing Hsp90 mutation E47A on Aha1 association. Experiments were carried out as described in b. e Y313E and E47A in Hsp90 additively promote Aha1 interaction. The triple mutant E47A/H210A/Y313E displays partially restored Aha1 interaction. Experiments were performed as above. Please see Supplementary Fig. 2a, b for more information. Source data for this figure are provided as a Source Data File

Fig. 3

Y313 phosphomimetic substitution affects conformation of all Hsp90 domains. Hydrogen exchange mass spectrometry shows global impact of Y313E phosphomimetic substitution on Hsp90 conformation. Purified human Hsp90α (wild-type or Hsp90-Y313E) was diluted 1:20 into D₂O buffer and incubated for 30 s at 30 °C in the absence of nucleotide. The reaction was quenched with ice cold quench buffer, the protein digested with immobilized pepsin, desalted and analyzed by liquid chromatography mass spectrometry (LC–MS). Deuteron incorporation into Hsp90-Y313E minus deuteron incorporation into wild-type Hsp90 is plotted for all peptic peptides (shown is the difference between each pair of means ± standard error for n = 3 independent experiments). For each peptide, bars depicting increased exchange for Hsp90-Y313E (compared to wild-type Hsp90) extend to the right, while bars showing decreased exchange for Hsp90-Y313E relative to wild-type Hsp90 extend to the left. Red lines indicate domain boundaries. CL, charged linker. The source data file contains the mean values ± SEM of deuteron incorporation for each corresponding peptide obtained from wild-type Hsp90 and Hsp90-Y313E, as well as the difference between the two sets of means ± SEM. The values underlying the means are no longer available

Fig. 4

Y313E-induced conformational changes probed by methyl-TROSY NMR. Selected regions from the overlay of the ¹H-¹³C-HMQC spectra of wild-type (black) and Hsp90-Y313E (red), showing signals that experience changes in chemical shifts and significant line broadening. The bottom panel includes an overlay of wild-type Hsp90 in the AMPPNP bound state (green) for I26 and I110 that undergo large chemical shift changes upon nucleotide addition due to Hsp90-N/N dimerization (see Supplementary Fig. 4). The Ile residues that experience significant chemical shift changes after phosphomimetic mutation mapped on the structure of Hsp90 in the closed state. One subunit is shown in gray, while in the other subunit N-, M- and C-domain of Hsp90 are colored blue, green, and orange, respectively. The site of the phosphomimetic mutation is shown in pink. Source data for this figure are provided as a Source Data File

Fig. 5

Modulation of Aha1–Hsp90 interaction by Y313E phosphomimetic substitution. Selected sets of signals from the titration of Y313E (left, black) or wild-type Hsp90 (right, black) with wild-type Aha1 (green), Aha1-N (cyan), or Aha1-C (pink). The lower panel shows the signal of an Ile residue from Hsp90-C for which no assignment is currently available (designated C6). Roman numbering denotes boxes extracted from the complete spectra shown in Supplementary Fig. 5

Fig. 6

Aha1-N and Aha1-C chemical shift perturbations mapped on the Hsp90 closed dimer. Aha1-N affected Hsp90 residues are shown in cyan and Aha1-C affected residues are shown in pink. The closed Hsp90 dimer is shown in dark and light gray, and the position of the Y313 phosphomimetic mutation is shown in yellow. The position of Aha1-N is modeled in complex with one of the two protomers. All Hsp90-C Ile residues are depicted in gray

Fig. 7

Hsp90–Y313E–Aha1 interaction profile shares similarities with Hsp90+ AMPPNP. Purified Hsp90α (wild-type or Y313E) and Aha1 proteins were mixed in the presence or absence of AMPPNP, followed by the addition of the crosslinking reagent BDP-NHP which crosslinks adjacent lysines (maximal cross-linking distance is 42.1 Å between the alpha-carbons of the linked lysine residues). Samples were then subjected to LC–MS analysis, crosslinked lysines were identified and the crosslink intensities were calculated and grouped for each crosslink pair. Error bars represent 95% confidence interval (n = 6 independent experiments). Please see Supplementary Fig. 3b, c for more information. Source data for this figure are provided as a Source Data File

Fig. 8

Enhanced Hsp90–Y313E–Aha1 interaction depends on Aha1-C binding to Hsp90-M domain. a Phosphomimetic substitution of Y313 in one protomer of Hsp90 is sufficient to stimulate Aha1 association. HA or FLAG tagged Hsp90, wild-type or Y313E, were co-expressed in 293A cells and sequentially immunoprecipitated with anti-HA and anti-FLAG antibody-linked resins. The first round of IP was performed with anti-HA antibody-linked resin; bound proteins were eluted with HA peptide and then loaded onto anti-FLAG antibody-linked resin. Proteins purified from the second round of IP were eluted with SDS-sample buffer and subjected to western blot analysis. Indicated proteins were detected with specific antibodies. b Hsp90 interaction with Aha1-N is abrogated by mutation in Hsp90-M while interaction with Aha1-C is compromised by mutation in both Hsp90-N and Hsp90-M. FLAG-tagged Aha1-N or Aha1-C was co-expressed with HA-tagged Hsp90, wild-type or with the indicated mutations. Aha1 domains were immunoprecipitated with mouse anti-FLAG antibody-linked resin, and Hsp90 was detected by western blot with rat anti-HA antibody. c FLAG or HA tagged Hsp90, wild-type or mutants, were co-expressed in 293A cells and sequentially immunoprecipitated and analyzed as in panel a. Y313E-stimulated association of full-length Aha1 with Hsp90 requires phosphomimetic mutation of Y313 on only one protomer, but requires ATP binding to Hsp90-N on both protomers, since D93A mutation on either protomer negates the impact of Y313E. Aha1 binding to Hsp90-Y313E is also disrupted by W320A mutation in cis (on the same protomer containing Y313E), but no disruption occurs when W320 mutation occurs in trans to Y313E. Source data for this figure are provided as a Source Data File

Fig. 9

Y313 phosphorylation recruits Aha1 to Hsp90. The model shown here focuses on early steps in the cycle as Hsp90 proceeds from a client-loaded open conformation to dissociation of the client-loading co-chaperone p60^HOP, and finally to recruitment of the ATPase-stimulating co-chaperone Aha1. An apo-Hsp90 dimer adopts the open conformation with the two N-domains un-engaged (a). Apo-Hsp90 can bind the co-chaperone p60^HOP to facilitate client transfer from Hsp70 (not shown in the cartoon) to Hsp90. Phosphorylation of Y313 in the Hsp90 middle domain induces a conformational change (b), weakening interaction with HOP and promoting recruitment of Aha1. Aha1 binds to the Hsp90 dimer while Hsp90 N-domains remain disassociated, with Aha1-N interacting with Hsp90-M of the un-phosphorylated Hsp90 protomer and Aha1-C interacting with Hsp90-M of the Hsp90 protomer bearing phosphorylated Y313 (c). Sumoylation of K191 in Hsp90-N in the presence of ATP promotes a repositioning of Aha1-C to engage the N-dimerized closed conformation that is required for activation of Hsp90 ATPase activity (d). Hydrolysis of ATP to ADP, and subsequent release of ADP, bias Hsp90 to re-occupy the open conformation, enabling it to re-enter the ATP-directed chaperone cycle