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

Stetz G, Verkhivker GM - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Computational and Data Sciences, Schmid College of Science and Technology, Chapman University, Orange, California, United States of America.

ABSTRACT
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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Related in: MedlinePlus

Free Energy Calculations of the Substrate Binding with the DnaK Structures.Binding free energies and alanine scanning of the NRLLLTG substrate-interacting residues with the ADP-DnaK (A) and ATP-DnaK (C). Computational alanine scanning employed MM-GBSA calculations to evaluate the effect of alanine mutations for the substrate binding site residues on binding affinity using MD trajectories of the nucleotide/substrate-bound WT structures. The protocol involved a systematic modification of the inhibitor-interacting residues to alanine by eliminating side-chain atoms beyond Cβ, and measuring the effect of each mutation on binding affinity. The close-up of the NRLLLTG substrate binding mode and interacting residues is shown for ADP-DnaK (B) and ATP-DnaK (D). The peptide is shown in atom-colored sticks and annotated. The substrate binding site residues from the SBD-β subdomain are shown in orange sticks and annotated.
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pone.0143752.g008: Free Energy Calculations of the Substrate Binding with the DnaK Structures.Binding free energies and alanine scanning of the NRLLLTG substrate-interacting residues with the ADP-DnaK (A) and ATP-DnaK (C). Computational alanine scanning employed MM-GBSA calculations to evaluate the effect of alanine mutations for the substrate binding site residues on binding affinity using MD trajectories of the nucleotide/substrate-bound WT structures. The protocol involved a systematic modification of the inhibitor-interacting residues to alanine by eliminating side-chain atoms beyond Cβ, and measuring the effect of each mutation on binding affinity. The close-up of the NRLLLTG substrate binding mode and interacting residues is shown for ADP-DnaK (B) and ATP-DnaK (D). The peptide is shown in atom-colored sticks and annotated. The substrate binding site residues from the SBD-β subdomain are shown in orange sticks and annotated.

Mentions: According to the crystallographic and NMR structure of the substrate-bound DnaK-SBD [19, 22], in the high affinity state the peptide binds in an extended conformation interacting with the SBD-β and SBD-α subdomains. The substrate binding mode and interactions are largely retained in the solution structure of complete DnaK construct complexed with [31]. We initiated simulations of the ADP/substrate-bound DnaK using the SBD-bound crystallographic conformation of the NRLLLTG peptide [19]. In the course of simulations, the substrate maintained the stable bound conformation locked in the crystallographic position through a dense network of van der Waals interactions and hydrogen bonds with the SBD domain (Fig 8A and 8B). The NRLLLTG peptide binding pocket is formed by residues from loops L1,2 and L3,4, but also stabilized by additional contacts with L4,5 and L5,6. The computed binding free energies registered an appreciable substrate affinity, also revealing the key contributions of I401, T403, M404, F426, A429, Q434, and V436 residues that could act as energetic hotspots of the substrate binding (Fig 8A). A significant role of these residues in determining binding energetics reflected substrate-induced stabilization of the L1,2 (residues 404–406) and L3,4 (residues 428–434) loops. These results are consistent with biochemical studies of substrate-DnaK binding in which M404A, V436F, and A429W mutants displayed > 10-fold loss of substrate binding affinity [38,39]. Mutagenesis studies have also shown that two single-site mutations, F426S and S427P could lead to the weakened peptide binding affinity [47]. Hence, binding free energy calculations reaffirmed that the substrate binding affinity may be predominantly determined by the interactions with specific hydrophobic residues in the L1,2 and L3,4 loops.


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

Stetz G, Verkhivker GM - PLoS ONE (2015)

Free Energy Calculations of the Substrate Binding with the DnaK Structures.Binding free energies and alanine scanning of the NRLLLTG substrate-interacting residues with the ADP-DnaK (A) and ATP-DnaK (C). Computational alanine scanning employed MM-GBSA calculations to evaluate the effect of alanine mutations for the substrate binding site residues on binding affinity using MD trajectories of the nucleotide/substrate-bound WT structures. The protocol involved a systematic modification of the inhibitor-interacting residues to alanine by eliminating side-chain atoms beyond Cβ, and measuring the effect of each mutation on binding affinity. The close-up of the NRLLLTG substrate binding mode and interacting residues is shown for ADP-DnaK (B) and ATP-DnaK (D). The peptide is shown in atom-colored sticks and annotated. The substrate binding site residues from the SBD-β subdomain are shown in orange sticks and annotated.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664246&req=5

pone.0143752.g008: Free Energy Calculations of the Substrate Binding with the DnaK Structures.Binding free energies and alanine scanning of the NRLLLTG substrate-interacting residues with the ADP-DnaK (A) and ATP-DnaK (C). Computational alanine scanning employed MM-GBSA calculations to evaluate the effect of alanine mutations for the substrate binding site residues on binding affinity using MD trajectories of the nucleotide/substrate-bound WT structures. The protocol involved a systematic modification of the inhibitor-interacting residues to alanine by eliminating side-chain atoms beyond Cβ, and measuring the effect of each mutation on binding affinity. The close-up of the NRLLLTG substrate binding mode and interacting residues is shown for ADP-DnaK (B) and ATP-DnaK (D). The peptide is shown in atom-colored sticks and annotated. The substrate binding site residues from the SBD-β subdomain are shown in orange sticks and annotated.
Mentions: According to the crystallographic and NMR structure of the substrate-bound DnaK-SBD [19, 22], in the high affinity state the peptide binds in an extended conformation interacting with the SBD-β and SBD-α subdomains. The substrate binding mode and interactions are largely retained in the solution structure of complete DnaK construct complexed with [31]. We initiated simulations of the ADP/substrate-bound DnaK using the SBD-bound crystallographic conformation of the NRLLLTG peptide [19]. In the course of simulations, the substrate maintained the stable bound conformation locked in the crystallographic position through a dense network of van der Waals interactions and hydrogen bonds with the SBD domain (Fig 8A and 8B). The NRLLLTG peptide binding pocket is formed by residues from loops L1,2 and L3,4, but also stabilized by additional contacts with L4,5 and L5,6. The computed binding free energies registered an appreciable substrate affinity, also revealing the key contributions of I401, T403, M404, F426, A429, Q434, and V436 residues that could act as energetic hotspots of the substrate binding (Fig 8A). A significant role of these residues in determining binding energetics reflected substrate-induced stabilization of the L1,2 (residues 404–406) and L3,4 (residues 428–434) loops. These results are consistent with biochemical studies of substrate-DnaK binding in which M404A, V436F, and A429W mutants displayed > 10-fold loss of substrate binding affinity [38,39]. Mutagenesis studies have also shown that two single-site mutations, F426S and S427P could lead to the weakened peptide binding affinity [47]. Hence, binding free energy calculations reaffirmed that the substrate binding affinity may be predominantly determined by the interactions with specific hydrophobic residues in the L1,2 and L3,4 loops.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Computational and Data Sciences, Schmid College of Science and Technology, Chapman University, Orange, California, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus