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Analysis of reconstituted tripartite complex supports avidity-based recruitment of Hsp70 by substrate bound J-domain protein

ABSTRACT: Hsp70 are ubiquitous, versatile molecular chaperones that cyclically interact with substrate protein(s). The initial step requires synergistic interaction of a substrate and a J-domain protein (JDP) cochaperone, via its J-domain, with Hsp70 to stimulate hydrolysis of its bound ATP. This hydrolysis drives conformational changes in Hsp70 that stabilize substrate binding. However, because of the transient nature of substrate and JDP interactions, this key step is not well understood. Here we leverage a well characterized Hsp70 system specialized for iron-sulfur cluster biogenesis, which like many systems, has a JDP that binds substrate on its own. Utilizing an ATPase-deficient Hsp70 variant, we isolated a Hsp70- JDP-substrate tripartite complex. Complex formation and stability depended on residues previously identified as essential for bipartite interactions: JDP-substrate, Hsp70-substrate and J-domain-Hsp70. Computational docking based on the established J-domain-Hsp70(ATP) interaction placed the substrate close to its predicted position in the peptide-binding cleft, with the JDP having the same architecture as when in a bipartite complex with substrate. Together, our results indicate that the structurally rigid JDP- substrate complex recruits Hsp70(ATP) via precise positioning of J-domain and substrate at their respective interaction sites - resulting in functionally high affinity (i.e., avidity). The exceptionally high avidity observed for this specialized system may be unusual because of the rigid architecture of its JDP and the additional JDP-Hsp70 interaction site uncovered in this study. However, functionally important avidity driven by JDP-substrate interactions is likely sufficient to explain synergistic ATPase stimulation and efficient substrate trapping in many Hsp70 systems.

INSTRUMENT(S): TripleTOF 5600, Synapt MS

ORGANISM(S): Escherichia Coli Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Igor Grochowina

LAB HEAD: Jaroslaw Marszalek

PROVIDER: PXD044739 | Pride | 2024-02-19

REPOSITORIES: Pride

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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

			Action	DRS
	HDX_DynamX_cluster_csv_output.zip	Other
	HDX_PLGS.zip	Other
	HDX_SEQ_RAW.zip	Other
	Hsc20_HDX_RAW.zip	Other
	Hsc20_Isu1_HDX_RAW.zip	Other

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Analysis of reconstituted tripartite complex supports avidity-based recruitment of Hsp70 by substrate bound J-domain protein

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