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Common functionally important motions of the nucleotide-binding domain of Hsp70.

ABSTRACT: The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate-binding domain (SBD) that binds client substrates, and the nucleotide-binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure-function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all-atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP- and ATP-unique classes, which reflect conformational trends that are unique to either the ADP- or ATP-bound states, respectively. "Mutual" class motions generally describe "in-plane" and/or "out-of-plane" (scissor-like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The "unique" class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the "unique" type, regions of enhanced mobility can be identified; these are termed "hot spots," and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide-binding pocket was also found to influence the dynamics of the NBD significantly.

SUBMITTER: Golas EI

PROVIDER: S-EPMC4300246 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Common functionally important motions of the nucleotide-binding domain of Hsp70.

Gołaś Ewa I EI Czaplewski Cezary C Scheraga Harold A HA Liwo Adam A

Proteins 20141218 2

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate-binding domain (SBD) that binds client substrates, and the nucleotide-binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure-function relationships that link the binding state of the NBD to its conformational behavior, we analy ...[more]

PMID: 25412765

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Project description:Human KCNH2 channels (hKCNH2, ether-à-go-go [EAG]-related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD), which is crucial for channel function. Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif, in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channel regulation by cyclic nucleotides. While there have been significant advancements in the structural and functional characterization of the CNBHD of KCNH channels, a high-resolution structure of the hKCNH2 intracellular complex has been missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the intrinsic ligand is preserved within the β-roll region. Moreover, measurements of small-angle x-ray scattering profile in solution, as well as comparison with a recent NMR analysis of hKCNH2, revealed high agreement with the crystallographic structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863) which was not previously resolved in the NMR and cryo-EM structures. Electrophysiological analysis of charge-reversal mutations revealed the bridge's crucial role in hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the intrinsic ligand and the maintenance of the intracellular complex interface.

Dataset Information

Common functionally important motions of the nucleotide-binding domain of Hsp70.

Publications

Common functionally important motions of the nucleotide-binding domain of Hsp70.

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