Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

Nicolaı̈, Adrien; Delarue, Patrice; Senet, Patrick

doi:10.1371/journal.pcbi.1003379

Cited by 31 publications

(48 citation statements)

References 116 publications

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“…To elucidate the link between ATP-induced domain docking, substrate binding, and intradomain flexibility of the βSBD, we used full-atom molecular dynamic (MD) simulations. As expected from previously reported attempts to monitor domain-docking/domain-undocking transitions in Hsp70s using full-atom MD simulations (29), no full transition between the βSBD end-point conformations (NBD-docked and -undocked) was observed in our MD simulations of the full-length DnaK. Thus, to monitor conformational transitions in the βSBD, we dissected key functional steps of the Hsp70 allosteric cycledissociation from the NBD, β8 rearrangement, and substrate binding-using truncations of functionally and structurally important regions that enable conformational transitions in the DnaK to occur on the MD timescale (Table S2).…”

Section: Resultssupporting

confidence: 64%

Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Zhuravleva

Gierasch

2015

Proc. Natl. Acad. Sci. U.S.A.

105

164

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Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and βSBD and releasing of the α-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the βSBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the βSBD and force disengagement of SBD strand β8 from strand β7, which leads to repacking of a βSBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire βSBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the βSBD-the NBD binding interface and the substrate-binding site-confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone-client interactions. molecular chaperone | protein quality control | NMR chemical shift perturbations | conformational selection | entropically driven allostery H eat shock protein 70 (Hsp70) molecular chaperones are central players in protein quality control systems for organisms from bacteria to humans (1, 2). All Hsp70s consist of two highly conserved domains: an N-terminal nucleotide-binding domain (NBD), which regulates the affinity of substrate binding, and a C-terminal substrate-binding domain (SBD), which binds to exposed hydrophobic stretches of client proteins and is made up of a β-sandwich domain (the βSBD), and an α-helical lid (the αLid) (Fig. 1A). Hsp70 functions rely on interdomain allostery: ATP hydrolysis in the NBD controls thermodynamics and kinetics of substrate binding and release; in turn, substrate binding to the SBD stimulates ATPase activity in the NBD (3-5).Mechanistic understanding of Hsp70 function has been greatly enhanced by recent breakthrough achievements in structural characterization of individual functional steps of the allosteric cycle for the Escherichia coli Hsp70 DnaK. New crystal structures and NMR analysis of DnaK have provided descriptions of three major functional states: ADP-bound (6, 7), 9), and ATP/substrate-bound (10), which have distinct arrangements of the four structural units NBD, βSBD, αLid, and interdomain linker (Fig. 1A). In the ADP-bound (domain-undocked, linker-unbound) state, the two DnaK domains, the NBD and SBD, behave independently, with the interdomain linker exposed to the solvent and ...

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Section: Resultssupporting

confidence: 64%

Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Zhuravleva

Gierasch

2015

Proc. Natl. Acad. Sci. U.S.A.

105

164

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“…These results are consistent with chemical shift perturbations data that detected rotational dynamics of subdomain IIB and partial opening of the nucleotide-binding cleft in the ADP-bound DnaK [ 32 ]. The relevance of subdomain IIB motions for DnaK allostery was stressed in various experiments [ 32 – 34 ] and computational studies [ 60 , 64 ]. Our simulations depicted a considerable heterogeneity of the lid conformations and the entire conformational ensemble of the ADP-DnaK ( S1 Fig ).…”

Section: Resultsmentioning

confidence: 99%

“…Conformational heterogeneity of Hsp70 was also evident in microsecond MD simulations of full-length human Hsp70 in explicit solvent, reproducing major conformational states observed in single-molecule Förster resonance energy transfer (FRET) experiments and small-angle X-ray scattering (SAXS) data of Hsp70 homologs [ 63 ]. Unbiased MD simulations and free energy landscape analysis were performed for Hsp70-DnaK in nucleotide-free and nucleotide-bound states, revealing potential conformational pathways and a significant number of mediating residues through which the ATP binding may propagate an allosteric signal to SBD [ 64 ]. MD simulations were also combined with mutagenesis, and enzymatic assays to identify functional residues that govern conformational motions in the apo, ATP-bound, and ADP-bound states of DnaK [ 65 ].…”

Section: Introductionmentioning

confidence: 99%

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

Stetz

Verkhivker

2015

PLoS ONE

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Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

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“…This is in line with observation made on the interaction of Hop and Hsp70 from other species [ 25 ]. It is known that the C-terminal domain of Hsp70 in which the EEVD motif is located maintains a similar conformation in the presence of ADP and in a nucleotide-free form of the chaperone [ 26 ]. Therefore, it is not surprising that nucleotide-free and ADP-bound forms of PfHsp70-1 interact with PfHop better than the ATP-bound form of the chaperone ( Fig 2 ; S2 Table ).…”

Section: Discussionmentioning

confidence: 99%

Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity

et al. 2015

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Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally eluted as a protein species of twice its predicted size, suggesting that it may occur as a dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition, the protein is capable of self-association. The findings suggest that PfHop serves as a module that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this study are important towards the development of possible antimalarial inhibitors targeting the cooperation of these two chaperones.

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Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

Cited by 31 publications

References 116 publications

Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Substrate-binding domain conformational dynamics mediate Hsp70 allostery

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity

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