Direct inter-subdomain interactions switch between the closed and open forms of the Hsp70 nucleotide-binding domain in the nucleotide-free state

Abstract

The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the ;closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.

Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70

“…32 It has been suggested previously that the closed conformation is apparently stabilized by the formation of a solvent exposed salt-bridge between glutamic acid-268 and lysine-56. 35 Intrigued by the formation of this intramolecular interaction, we then sought to generate a co-crystal structure with the bacterial natural product sangivamycin 10 ( K D = 3.3 μM) (Figure 4). …”

Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

“…ATP complex was built by substituting ADP with ATP-Mg 2+ coordinates obtained from 2EA8 structure [44]. Hsp110 X-ray structure of S. cerevisiae (PDB ID: 3C7N_A [12]) in complex with ATP-Mg 2+ was utilized as starting point.…”

A Small Molecule Inhibitor of ATPase Activity of HSP70 Induces Apoptosis and Has Antitumor Activities

“…The NMR data yielded a range of conformations of this substance ( Figures S2C and S2D), which were then subjected to computational analysis to elucidate the low energy conformation of free Az ( Figure S2E). The conformational information and STD NMR data were utilized for molecular docking studies with HSP70 (PDB code 4IO8, a VER-155008 complex form; and PDB code 2E8A, an AMP-PNP [adenylyl-imidodiphosphate, a non-hydrolyzable analog of ATP] bound form) to obtain a more detailed view of the binding mode of Az to HSP70 (Cho et al, 2011;Schlecht et al, 2013;Shida et al, 2010). The results of a molecular modeling study show that Az adopts a conformation that is closely overlaid with AMP-PNP in the ATP binding site of HSP70 ( Figure 2A).…”