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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

The Protein Stability Analysis of DnaK Structures.Protein stability changes ΔΔG are computed using a systematic alanine scanning. The protocol involved modification of the protein residues to alanine and computing the effect of each mutation on protein stability using FoldX (A,B) and DUET (C,D) methods respectively. The profiles are annotated using residue numbering in the solution structure of the ADP-bound DnaK (pdb id 2KHO) for (A, C) and the crystal structure of an ATP-bound DnaK (pdb id 4B9Q) for (B, D). The profiles are shown as bars colored according to the adopted scheme: IA (in blue), IB (in red), IIA (in green), IIB (in cyan), the inter-domain linker (in black), SBD-α (in magenta), and SBD-β (in orange). The inset shows protein stability changes of the SBD-β residues (orange-colored lines with marron-colored filled squares).
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pone.0143752.g006: The Protein Stability Analysis of DnaK Structures.Protein stability changes ΔΔG are computed using a systematic alanine scanning. The protocol involved modification of the protein residues to alanine and computing the effect of each mutation on protein stability using FoldX (A,B) and DUET (C,D) methods respectively. The profiles are annotated using residue numbering in the solution structure of the ADP-bound DnaK (pdb id 2KHO) for (A, C) and the crystal structure of an ATP-bound DnaK (pdb id 4B9Q) for (B, D). The profiles are shown as bars colored according to the adopted scheme: IA (in blue), IB (in red), IIA (in green), IIB (in cyan), the inter-domain linker (in black), SBD-α (in magenta), and SBD-β (in orange). The inset shows protein stability changes of the SBD-β residues (orange-colored lines with marron-colored filled squares).

Mentions: We analyzed allosteric interactions in the DnaK structures from two different but complementary points of view. According to a mechanistic-based model, allosteric mechanism is described as nucleotide-induced signal propagation from one site to another via a series of conformational changes and intermediate structures. In the ensemble-based model of allostery [104–107], ligand binding or mutations capable of altering the energetic hierarchy of states in the thermodynamic ensemble can change the allosteric coupling between two allosteric sites, even when the network of allosteric residues that physically connects these two sites remains largely unaffected. To explore the energetic determinants of the ensemble-based allostery, we computed protein stability changes by using a systematic alanine scanning of the DnaK structures, in which the free energy changes were averaged over MD-based conformational ensembles. Of particular interest was to quantify the energetics of allosteric coupling between the NBD-SBD interface and highly flexible substrate binding loops in the ATP-DnaK, which is arguably central to the allosteric structural changes [37]. We also examined how fast substrate binding in the ATP-DnaK could control energetics of allosteric interactions to ensure efficient transmission of the substrate signal to the NBD-SBD interface and the nucleotide-binding site. The protein stability changes in DnaK structures were computed using the FoldX approach [123,124]. In a systematic alanine scanning of DnaK residues we utilized a graphical user interface for the FoldX force field calculations [125] that was implemented as a plugin for the YASARA molecular graphics suite [126]. If a free energy change between a mutant and the wild type (WT) proteins ΔΔG = ΔG (MT)-ΔG (WT) > 0, the mutation is destabilizing, while when ΔΔG <0 the respective mutation is stabilizing. FoldX could be sensitive to conformational changes in the MD ensembles, and the crystal structure is typically more suitable for stability predictions that any single snapshot. To ensure reproducibility of the free energy computations and still consider functionally important dynamic changes, we computed the average ΔΔG values using multiple samples (~100–200) from stable equilibrium ensembles using a modified FoldX protocol [127,128]. Moderate destabilizing free energy changes were distributed across all subdomains, with a relatively small number of sites producing a significant destabilizing effect upon mutation (ΔΔG >1.5–2.0 kcal/mol) (Fig 6A and 6B). Moreover, the pattern of protein stability changes in the open and closed DnaK structures was quite similar, which is consistent with similar free energies associated with these allosteric states [44]. These results are consistent with the experimental observations in which melting temperatures of the WT DnaK and a number of important DnaK mutants were rather similar [52], indicating that even mutations of functionally critical for allosteric communication residues could not cause global structural defects and markedly impair stability of the protein fold. Noteworthy, however, mutations of allosterically important residues in the NBD (K71, E171, P143, Y145, F146, D148A, R151, K155 R167, I168) and SBD-β (I438, V440, L454, L484) may still incur a moderate but noticeable destabilizing effect (Fig 6A and 6B). Accordingly, structural perturbations exerted on these residues could redistribute local conformational ensembles and alter energetics of functional regions that are involved in ligand-based allostery. Notably, these changes would not compromise structural integrity of DnaK folds or significantly affect the total energy, as the ligand-induced cooperativity is different from cooperativity related to folding/unfolding transitions. We also computed the protein stability changes (Fig 6C and 6D) using an alternative computational approach DUET [129]. This machine learning method produces a consensus prediction by integrating the results of mutation Cutoff Scanning Matrix (mCSM) algorithm that relies on structural signatures of protein residue environments [130] and Site Directed Mutator (SDM) method that is based on a statistical potential energy function [131]. Despite differences between the FoldX and DUET approaches, these methods revealed comparable protein stability changes, though DUET predictions resulted in the larger free energy differences. More importantly, both approaches displayed similar patterns of protein stability changes in the DnaK regions.


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)

The Protein Stability Analysis of DnaK Structures.Protein stability changes ΔΔG are computed using a systematic alanine scanning. The protocol involved modification of the protein residues to alanine and computing the effect of each mutation on protein stability using FoldX (A,B) and DUET (C,D) methods respectively. The profiles are annotated using residue numbering in the solution structure of the ADP-bound DnaK (pdb id 2KHO) for (A, C) and the crystal structure of an ATP-bound DnaK (pdb id 4B9Q) for (B, D). The profiles are shown as bars colored according to the adopted scheme: IA (in blue), IB (in red), IIA (in green), IIB (in cyan), the inter-domain linker (in black), SBD-α (in magenta), and SBD-β (in orange). The inset shows protein stability changes of the SBD-β residues (orange-colored lines with marron-colored filled squares).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143752.g006: The Protein Stability Analysis of DnaK Structures.Protein stability changes ΔΔG are computed using a systematic alanine scanning. The protocol involved modification of the protein residues to alanine and computing the effect of each mutation on protein stability using FoldX (A,B) and DUET (C,D) methods respectively. The profiles are annotated using residue numbering in the solution structure of the ADP-bound DnaK (pdb id 2KHO) for (A, C) and the crystal structure of an ATP-bound DnaK (pdb id 4B9Q) for (B, D). The profiles are shown as bars colored according to the adopted scheme: IA (in blue), IB (in red), IIA (in green), IIB (in cyan), the inter-domain linker (in black), SBD-α (in magenta), and SBD-β (in orange). The inset shows protein stability changes of the SBD-β residues (orange-colored lines with marron-colored filled squares).
Mentions: We analyzed allosteric interactions in the DnaK structures from two different but complementary points of view. According to a mechanistic-based model, allosteric mechanism is described as nucleotide-induced signal propagation from one site to another via a series of conformational changes and intermediate structures. In the ensemble-based model of allostery [104–107], ligand binding or mutations capable of altering the energetic hierarchy of states in the thermodynamic ensemble can change the allosteric coupling between two allosteric sites, even when the network of allosteric residues that physically connects these two sites remains largely unaffected. To explore the energetic determinants of the ensemble-based allostery, we computed protein stability changes by using a systematic alanine scanning of the DnaK structures, in which the free energy changes were averaged over MD-based conformational ensembles. Of particular interest was to quantify the energetics of allosteric coupling between the NBD-SBD interface and highly flexible substrate binding loops in the ATP-DnaK, which is arguably central to the allosteric structural changes [37]. We also examined how fast substrate binding in the ATP-DnaK could control energetics of allosteric interactions to ensure efficient transmission of the substrate signal to the NBD-SBD interface and the nucleotide-binding site. The protein stability changes in DnaK structures were computed using the FoldX approach [123,124]. In a systematic alanine scanning of DnaK residues we utilized a graphical user interface for the FoldX force field calculations [125] that was implemented as a plugin for the YASARA molecular graphics suite [126]. If a free energy change between a mutant and the wild type (WT) proteins ΔΔG = ΔG (MT)-ΔG (WT) > 0, the mutation is destabilizing, while when ΔΔG <0 the respective mutation is stabilizing. FoldX could be sensitive to conformational changes in the MD ensembles, and the crystal structure is typically more suitable for stability predictions that any single snapshot. To ensure reproducibility of the free energy computations and still consider functionally important dynamic changes, we computed the average ΔΔG values using multiple samples (~100–200) from stable equilibrium ensembles using a modified FoldX protocol [127,128]. Moderate destabilizing free energy changes were distributed across all subdomains, with a relatively small number of sites producing a significant destabilizing effect upon mutation (ΔΔG >1.5–2.0 kcal/mol) (Fig 6A and 6B). Moreover, the pattern of protein stability changes in the open and closed DnaK structures was quite similar, which is consistent with similar free energies associated with these allosteric states [44]. These results are consistent with the experimental observations in which melting temperatures of the WT DnaK and a number of important DnaK mutants were rather similar [52], indicating that even mutations of functionally critical for allosteric communication residues could not cause global structural defects and markedly impair stability of the protein fold. Noteworthy, however, mutations of allosterically important residues in the NBD (K71, E171, P143, Y145, F146, D148A, R151, K155 R167, I168) and SBD-β (I438, V440, L454, L484) may still incur a moderate but noticeable destabilizing effect (Fig 6A and 6B). Accordingly, structural perturbations exerted on these residues could redistribute local conformational ensembles and alter energetics of functional regions that are involved in ligand-based allostery. Notably, these changes would not compromise structural integrity of DnaK folds or significantly affect the total energy, as the ligand-induced cooperativity is different from cooperativity related to folding/unfolding transitions. We also computed the protein stability changes (Fig 6C and 6D) using an alternative computational approach DUET [129]. This machine learning method produces a consensus prediction by integrating the results of mutation Cutoff Scanning Matrix (mCSM) algorithm that relies on structural signatures of protein residue environments [130] and Site Directed Mutator (SDM) method that is based on a statistical potential energy function [131]. Despite differences between the FoldX and DUET approaches, these methods revealed comparable protein stability changes, though DUET predictions resulted in the larger free energy differences. More importantly, both approaches displayed similar patterns of protein stability changes in the DnaK regions.

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