Computational Analysis of Residue Interaction Networks and Coevolutionary Relationships in the Hsp70 Chaperones: A Community-Hopping Model of Allosteric Regulation and Communication
[['The evolutionary conserved and versatile 70-kilodalton ( kDa ) heat shock proteins Hsp70s play a central role in supervision of various protein folding processes and coordination of a broad range of cellular events–from maintenance of cellular homeostasis to regulation of the heat shock response [1–8] .', 'The Hsp70 proteins cooperate with chaperones of other families to facilitate protein folding , prevent aggregation , and ensure stabilization and quality control of native regulatory proteins [9–12] .', 'The Hsp70 biochemical cycle involves a precisely orchestrated succession of nucleotide-induced allosteric structural changes that are executed through complex and adaptive interactions with co-chaperones , particularly J-domain proteins DnaJ and Hsp40 which accelerate ATP hydrolysis , and nucleotide exchange factors assisting in a timely progression of the ATP-ADP exchange [13–16] .', 'During this cycle , ATP binding in the nucleotide-binding domain ( NBD ) accelerates substrate dissociation from the substrate-binding domain ( SBD ) , while substrate binding synchronously stimulates ATPase hydrolysis in the NBD ( Fig 1 ) .', 'Structural and biophysical investigations of various E . coli Hsp70 ( DnaK ) constructs [17–26] have established that the NBD and SBD are only loosely coupled in the extended ADP-bound and the nucleotide-free states , whereas ATP binding shifts thermodynamic preferences and stabilizes a compact domain-docked DnaK structure , leading to stimulation of the ATPase activity and substrate release ( Fig 1 ) .', 'The solution NMR structures of the ADP-DnaK and apo DnaK ( Figs 1 and 2 ) have confirmed that the NBD and SBD could only weakly associate in the domain-undocked state of the chaperone [27] .', 'Chemical-shift perturbation analysis of the DnaK states has provided evidence of ATP-induced rotational movements of the NBD subdomains that promote binding of the inter-domain linker and stabilization of the NBD-SBD interface [28] .', 'The crystal structures of the ATP-bound DnaK [29 , 30] have revealed a synchronous docking of the SBD-β and SBD-α subdomains to the NBD in the ATP-DnaK , that causes accelerated substrate dissociation ( Figs 1 and 2 ) .', 'The early biochemical studies [31] and subsequent electron spectroscopy experiments [32–35] have established the existence of multiple conformational states in the Hsp70 proteins , particularly showing that nucleotide exchange could promote formation of partially undocked meta-stable intermediates .', 'Recent NMR studies have discovered that dynamic changes in the inter-domain and substrate binding regions are coupled and may coordinate ATP hydrolysis and substrate release via an entropy-driven allosteric mechanism [36] .', 'While the ADP-DnaK structure with high substrate affinity may be stabilized by both enthalpy and entropy contributions , the thermodynamics of the ATP-bound DnaK with low substrate affinity may be mainly driven by entropy changes [37] .', 'The crystallographic and NMR studies of the yeast Hsp110 ( Sse1 ) [38–41] have revealed that the ATP-bound state can adopt a similar docked conformation , but ATP hydrolysis in Sse1 may proceed without triggering any appreciable conformational changes ( Fig 2 ) .', 'Consequently , a limited entropy-driven allostery could present a dominant “modus operandi” of the Sse1 biochemical cycle , where ATP hydrolysis is coupled to the substrate release via redistribution of conformational dynamics in the functional regions .', 'Mutagenesis studies have quantified the contributions of functional residues to allosteric signaling and inter-domain communications of DnaK [42–48] .', 'Several mutational variants of the NBD residues could maintain ATP binding and hydrolysis functions although they are severely deficient in allosteric signaling: Y145A , N147A , and D148A [42] , P143G and R151A [43] , K155D , R167D [44] and D326V [47] .', 'Important functional sites were also found in the SBD regions , where mutations K414I [45] , P419A [46] , and N415G [47] may completely abolish or significantly weaken allosteric interactions in the DnaK chaperone .', 'Mutations in the inter-domain linker region of DnaK could shut down functional activity of the DnaK-DnaJ chaperone systems [48] .', 'Mutagenesis and functional experiments of DnaK have determined the role of the SBD-α lid helices and the hydrophobic regions of the SBD-β in coordinating substrate binding affinity and kinetics of substrate dissociation [49–52] .', 'Recent breakthrough studies have discovered the previously overlooked divergence of allosteric signaling pathways in the DnaK chaperone by convincingly demonstrating how mutations of regulatory sites ( I438A , V440A , L484A , D481A , D481K ) could selectively interfere with direction-dependent steps of allosteric communication [53] .', 'According to this seminal work , functional cycle in DnaK may be allosterically controlled through concerted rearrangements of the specific inter-domain interactions that couple nucleotide exchange with substrate binding and release .', 'Mechanisms of allosteric signaling are ultimately determined by the thermodynamics of a system that can be described using ensemble-based computational models of allosteric interactions [54–56] .', 'Computational studies have investigated various molecular factors underlying allosteric regulatory mechanisms in the DnaK chaperones by simulating dynamics and energetics of the crystal structures and allosteric pathways [57–68] .', 'Molecular dynamics ( MD ) simulations and elastic network models have explored evolution and dynamics of the Hsp70 chaperones in binding with client proteins [57] and molecular aspects of nucleotide-induced conformational transitions in these chaperones [58] .', 'The diversity of conformational sates observed in biophysical experiments of the human Hsp70 has been successfully reproduced in atomistic simulations [60] .', 'The free energy landscape mapping of the DnaK structures has also suggested several mediating residues that may be instrumental in signal propagation between the NBD and SBD regions [61] .', 'Biophysical simulations and mutagenesis experiments have characterized several hinge residues controlling the nucleotide-dependent allostery in DnaK [62] .', 'Functional assays combined with perturbation response scanning analysis have identified a group of regulatory residues in subdomain IA of the NBD that promote allosteric interactions and inter-domain signal transmission [64] .', 'According to this study , while allosteric coupling in Hsp70 proteins could be maintained through clusters of conserved interactions , binding to co-chaperones may be facilitated by coevolving flexible residues in the subdomain IIA .', 'Coevolutionary analysis has allowed to capture large-scale conformational transformations of the Hsp70 chaperones and predicted functional dimer interactions between Hsp70 proteins [66] .', 'Computational modeling of the residue interactions in combination with in silico alanine scanning of the Hsp70 residues has probed molecular determinants and specific role of functional sites in allosteric signaling and biochemical cycle [67] .', 'MD simulations have elucidated the molecular determinants underlying ligand-induced modulation of conformational dynamics in the DnaK structures , showing that local dynamics changes in response to ligand binding may be coupled to allosteric structural rearrangements [68] .', 'The relationships between protein dynamics and allosteric signaling can be conveniently explored using structural analysis of the residue interaction networks [69–71] .', 'This approach can successfully identify functional residues [72 , 73] , describe ligand-induced shifts in conformational populations of allosteric proteins [74–78] and reconstruct allosteric communication pathways [79] .', 'MD simulations and network modeling have been combined to explore conformational ensembles of the Hsp70 chaperones [80] .', 'In our most recent investigation , dynamics-based network modeling and community detection approaches have joined forces in probing mechanisms of allosteric inhibition in the DnaK and human Hsp70 proteins [81] .', 'According to this study , functional effects of allosteric modulators may be linked with the inhibition of specific interaction networks that alter structural environment of the regulatory sites .', 'Despite advances in the experimental and computational studies of the Hsp70 mechanisms , the role and contribution of functional residues and specific interactions implicated in allosteric regulation are yet to be fully understood and properly quantified .', 'Moreover , the outstanding questions concerning modular organization of the allosteric interaction networks and hierarchy of interactions that control functional cycle of the Hsp70 chaperones remained largely unexplored .', 'Current understanding of allosteric communication pathways in the Hsp70 chaperones remains mostly mechanistic , lacking a proper physical foundation and atomistic insights that are required to rationalize latest experimental data on direction-specific pathways of allosteric regulation in DnaK [53] .', 'In this work , we employed a computational framework that combined atomistic and coarse-grained simulations of the Hsp70 structures with coevolutionary analysis and network-centric modeling .', 'The goal of this study was to elucidate in a systematic manner dynamic and evolutionary factors underlying allosteric structural transformations of the Hsp70 proteins .', 'A novel methodological aspect of this work was the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction and analysis of allosteric interaction networks .', 'Statistical coupling analysis [82–84] , mutual information ( MI ) model [85–87] and other evolutionary approaches [88–91] have shown that functionally significant residues can be connected via coevolutionary relationships .', 'Recent studies have revealed the important role of coevolving residues in mediating residue-residue contacts [92] , protein folding [93] and protein-protein recognition [94] .', 'Coevolving residues are often close to each other in the protein structure [95 , 96] and may form independent structural modules ( or protein sectors ) with distinct biochemical functions [97 , 98] .', 'Networks of residues with high mutual information can also characterize structural proximity of functionally important sites [99 , 100] .', 'In the current study , we investigate the relationships between conformational dynamics , coevolutionary residue associations and hierarchy of allosteric interactions in the Hsp70 proteins .', 'By performing a community decomposition of residue interactions , we find that regulatory sites can be distinguished by their high network centrality and integrating role in modular organization of allosteric interactions .', 'We show that the inter-domain communities may be coordinated by key functional centers that are surrounded by coevolving flexible residues in order to facilitate conformational transitions .', 'Cooperative rearrangements in these communities can adequately describe allosteric changes and population shifts in conformational ensembles of the Hsp70 proteins .', 'By using allosteric residue propensities , we introduce a community-hopping model of communication pathways that explained the asymmetry of allosteric control in the DnaK chaperone .', 'The results of this study reconcile a wide spectrum of functional experiments by providing a network-centric outlook of the conformational dynamics , coevolution and interaction networks in the Hsp70 proteins .', 'We argue that these factors may act as synchronizing forces that shape up the efficiency and robustness of allosteric regulatory mechanisms in these chaperones .'], ['We began by investigating the relationships between conformational dynamics , sequence conservation and residue coevolution in the Hsp70 protein family .', 'MD simulations were independently performed for full-length two-domain Hsp70 structures: DnaK_GEOKA ( pdb is 2V7Y ) DNAK_ECOLI ( pdb id 2KHO , 4B9Q , 4JNE , 4JN4 ) , HS7C_BOVIN ( pdb id 1YUW , 4FL9 ) , and HSP7F_YEAST ( Hsp homolog Sse1 ) proteins ( pdb id 2QXL , 3C7N , 3D2E , 3D2F ) .', '( Fig 2 ) .', 'We first analyzed evolutionary factors underlying allosteric regulation of the DnaK chaperone .', 'By analyzing the sequence conservation profile ( Fig 3A ) we identified evolutionary features that may differentiate regulatory sites of the DnaK chaperone from nonfunctional conserved residues .', 'The highly conserved residues were primarily assembled in the core regions of subdomains IA , IIA , IIB and the SBD-β subdomain .', 'Functional residues involved in the nucleotide binding and allosteric regulation were highly conserved ( K70 , R71 , P143 , Y145 , F146 , R151 , E171 , D393 , and V396 ) , while several other regulatory sites ( D148 , K155 , R167 , I168 , K414 , D481 ) could experience small conservative modifications during evolution .', 'Using MISTIC approach [99 , 100] we also characterized the extent of mutual information and coevolutionary couplings between residue pairs in the Hsp70 proteins .', 'Coevolution of protein residues may emerge from different factors including phylogeny-driven preferences for compensatory substitutions and structural constraints imposed by protein stability , adaptability to binding partners and regulatory functions .', 'In particular , coevolutionary signals of highly conserved residues in the protein core that undergo a small number of independent changes can be often overestimated , while correlated changes arising from molecular coevolution may be inadvertently suppressed [101–103] .', 'To discriminate coevolutionary associations driven by functional constraints from those determined by common ancestry , the covariance metric based on MI calculations was adjusted by the average product correction ( APC ) [104–106] .', 'Based on computations of coevolutionary residue matrices , we assembled a network of coevolutionary coupled residues in which the links between nodes ( residues ) represented mutual information shared by the respective residues .', 'We then estimated cumulative mutual information ( cMI ) and ensemble-based proximity mutual information ( pMI ) residue profiles of the DnaK structures .', 'A considerable spread in the cMI scores of functional residues was observed , particularly in the NBD subdomain IA and SBD-β regions , suggesting that cumulative coevolutionary score may not be a strong differentiating feature of the regulatory centers ( Fig 3B ) .', 'Functional residues involved in DnaK allostery could be much better distinguished by their high pMI values ( Fig 3C and 3D ) , revealing strong propensities of regulatory sites to be surrounded by clusters of highly coevolving residues in the protein structure .', 'Of particular interest were high pMI scores of key residues that communicate signal of the ATPase hydrolysis from the NBD region ( K70 , R71 , Y145 , F146 , R151 , R167 , I168 , E171 ) , to the inter-domain interface ( K414 , D481 ) , and the SBD-β allosteric hotspot ( L454 , L484 ) [36 , 53] .', 'Conformational dynamics of the DnaK chaperone revealed several important trends and determined specific groups of residues that can be distinguished by their unique dynamic and coevolutionary signatures ( Fig 4 ) .', 'Structurally stable regions in the ADP-DnaK and ATP-DnaK forms included residue segments 69–73 and 140–171 from the subdomain IA that featured catalytic residues K70 , E171 and conserved regulatory residues P143 , F146 , D148 , R151 , K155 , R167 , and I168 ( Fig 4A and 4B ) .', 'To examine collective dynamics and identify hinge sites of global motions in the DnaK structures , we supplemented MD simulations by coarse-grained Gaussian network ( GNM ) analysis [107–109] .', 'The GNM-based residue fluctuations averaged over the three lowest frequency modes were analyzed to characterize functional dynamics of the DnaK structures .', 'The local maxima in these profiles correspond to flexible regions undergoing global structural changes , while the local minima are typically attributed to structurally rigid sites that serve as hinge points that coordinate collective dynamics .', 'In the ADP-DnaK , the major hinge site ( D385 , V386 ) was located in the region connecting the subdomain IA of the NBD with the inter-domain linker ( Fig 4C ) .', 'Among other local minima were residues 199–202 ( the inter-domain interface between IA and IIA subdomains ) and regulatory sites P419 and D481 ( SBD-β ) that bridge the inter-domain linker with the SBD-β subdomain .', 'The slow mode profile of the ATP-DnaK structure showed that binding site residues ( K70 , R71 ) and regulatory sites ( P143 , Y145 , F146 , R151 , K155 , R167 , I168 , T221 , P419 , I438 , V440 , and L454 ) were largely immobilized in collective motions and could form hinge centers ( Fig 4D ) .', 'These rigid residues were also evolutionary conserved and featured high pMI scores ( top 5% ) .', 'Hence , an important category of functional sites involved in allosteric regulation of DnaK may be characterized by structural stability , proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues .', 'We argue that these specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues .', 'Structural mapping of high pMI sites showed that these residues occupy stable regions in the DnaK structures and could form local clusters near the nucleotide binding site and in the SBD-β subdomain ( Fig 5A and 5B ) .', 'Several highly coevolving stable residues ( D194 , L195 , T199 , D201 ) were previously implicated in stimulation of the ATPase activity and form a local cluster in the subdomain IIA ( Fig 5C and 5D ) .', 'Structural proximity of these residues to the catalytic center E171 and nucleotide binding site may impose limitations on conformational diversity of coevolving residues in this region , which may explain the observed stability .', 'However , the vast majority of residues with high cMI scores tend to occupy flexible regions , primarily in the subdomain II of the NBD and the substrate binding region ( Fig 5C and 5D ) .', 'These observations corroborated with the notion that coevolving flexible residues undergoing cooperative structural changes may constitute recognition elements of substrate binding sites [110 , 111] .', 'Another group of regulatory Hsp70 residues ( D326 , K414 , N415 , Q442 and D481 ) revealed high cMI scores and intermediate mobility level ( Figs 4 and 5 ) .', 'Several coevolving flexible sites in subdomain IIA ( D211 , E217 , V218 ) have been implicated in DnaJ recognition [112 , 113] .', 'According to these studies , DnaJ domain interacts with DnaK loop ( residue 206–221 ) and mutations of residues D208 , E209 , D211 , 217 , and V218 on DnaK interfere with DnaK-DnaJ binding and stimulation of the ATPase activity .', 'A strong coevolutionary signal of these residues was previously noticed in a related computational study [64] , suggesting that binding of the subdomain IIA to co-chaperones of the J-domain family may be meditated by a cluster of highly coevolving and structurally proximal residues .', 'Notably , many single mutations and modifications of residue segments in the NBD regions can affect DnaJ binding or compromise ATPase stimulation ( residues Y146-D148 , R151 , D388 , D393 , R167 , N170 , T173 , E217-V218 , V388-L392 , L390-L391 ) [113] .', 'We found that these residues exhibited a significant coevolutionary signal , confirming that several chaperone functions , including co-chaperone recruitment , regulation of the ATPase activity and allosteric control , may be mediated through allosteric coupling of coevolving residues .', 'By consolidating conformational mobility profiles and coevolutionary residue propensities over all simulated Hsp70 proteins , we evaluated the extent of correlation between these parameters ( S2 Fig ) .', 'An appreciable correlation was observed between residue mobility and pMI scores at the intermediate levels of conformational flexibility .', 'Residues with small pMI values were associated with low sequence conservation and may be accompanied by the increased conformational mobility , but this trend does not take effect until an intermediate mobility level is reached .', 'At the same time , no correlation was found between conformational mobility and cMI scores ( S2 Fig ) .', 'While high pMI sites in the Hsp70 structures corresponded mostly to structurally stable residues , coevolving positions exhibited a wide range of conformational mobility with the peaks pointing to the middle part of the spectrum .', 'These findings corroborated with evolutionary studies of protein dynamics [110 , 111] suggesting that highly coevolving residues may preferentially occupy regions of intermediate mobility .', 'We argue that conformational variability of highly coevolving residues that surround rigid regulatory sites may enable concerted rearrangements of specific interactions associated with global allosteric changes .', 'Although crystal structures of the ATP-DnaK and ATP-Sse1 chaperones are similar , their dynamics was somewhat different .', 'MD simulations of the ATP-bound Sse1 structures produced fluctuation profiles that were exemplified by structural stability of the NBD residues , the inter-domain interface and the SBD-α region ( S3 Fig ) .', 'At the same time , we noticed the increased conformational mobility of the substrate binding region in the SBD-β subdomain .', 'These unique dynamic characteristics of the ATP-Sse1 structures resulted in the reduced number of local hinge sites that produce a smaller allosteric network with fewer mediating centers ( S3 Fig ) .', 'In the Sse1 structures , only few high cMI residues occupied flexible regions in the SBD-β , which may also contribute to a limited allostery in this chaperone .', 'We integrated coevolutionary analysis into construction and analysis of the residue interaction networks to test our hypothesis that dynamic and coevolutionary residue correlations may act as synchronizing forces to enable efficient and robust allosteric regulation .', 'In this model , the network edges ( interactions ) are weighted based on both dynamic and coevolutionary residue correlations that determine the shortest communication paths between residue nodes .', 'Residue centrality ( residue betweenness ) is a global network parameter that was computed to determine highly connected nodes in a global interaction network .', 'A propensity of protein residues to serve as global mediating centers of allosteric interaction networks was evaluated by considering common peaks in the residue centrality and structure-based pMI profiles .', 'We show that due to their unique networking and coevolutionary signatures these sites may control allosteric signaling and structural transformations during the Hsp70 functional cycle .', 'A strong relationship was found between high centrality and functional significance of DnaK residues .', 'Importantly , coevolutionary pMI scores ( Fig 3C and 3D ) and residue centrality profiles of the DnaK structures ( Fig 6A and 6B ) showed similar shapes , with regulatory sites mapped almost precisely onto the major peaks of these distributions .', 'In the ADP-bound DnaK , three major broad peaks corresponded to a residue cluster in the subdomain IA ( residues 140–151 ) , the linker region , and residues 479–482 ( L6 , 7 loop ) ( Fig 6A ) .', 'In the ATP-DnaK , the distribution peaks corresponded to the subdomain IA residues ( 140–154 , 161–175 ) , the SBD-β residues ( L454 , F476 , L484 ) and the inter-domain residues from loops L2 , 3 ( residues 412–420 ) , L4 , 5 loop ( 442-QGE-444 ) and L6 , 7 loop ( residues D481 , G482 ) ( Fig 6B ) .', 'Among major peaks were the nucleotide binding site residues ( K70 , R71 ) , and functional residues of allosteric communication located at the inter-domain regions ( R151 , K155 , R167 , I168 , K414 , N415 ) .', 'Structural mapping of functional sites showed that high centrality/high pMI sites R151 , K155 , R167 , I168 are interconnected and linked with a flexible SBD-β “arm” D481 at one side of the inter-domain interface ( Fig 6C and 6D ) .', 'Another inter-domain juncture is formed through specific interactions between highly coevolving functional residues K414 , N415 ( L2 , 3 loop of the SBD-β ) and D326 from subdomain IIA that reside in structural proximity of high pMI residue T221 .', 'Hence , the major inter-domain bridges may be established through coupling of coevolving functional residues that reside in local proximity of high centrality hinge centers .', 'Global network centrality and local proximity to coevolving interfacial residues may facilitate cross-talk between functional hinge centers in coordination of allosteric changes .', 'Mutational variants Y145A and D148A [42] , P143G and R151A [43] , K155D and R167D [44] , K414I [45 , 53] , D326V and N415G [47] may dramatically reduce or eliminate allosteric signaling in DnaK .', 'In light of our results , the loss of regulatory function may be determined not only by disruption of specific inter-domain contacts , but also by global alterations in the network connectivity leading to the reduced efficiency of allosteric interactions .', 'We argue that high network centrality and strong coevolutionary associations of regulatory sites may cause even minor mutations at these positions to be highly detrimental for allosteric regulation .', 'By aggregating coevolutionary residue scores and residue centrality profiles from equilibrium ensembles of all simulated Hsp70 proteins , we evaluated the relationship between these parameters .', 'There was only little correlation between residue centrality and cMI scores ( Fig 7A ) .', 'However , an appreciable correlation was found between residue centrality and pMI scores .', 'Furthermore , functional residues of DnaK regulation displayed consistently high coevolutionary and network centrality scores that were strongly correlated ( Fig 7B ) .', 'We extended this analysis by considering experimentally known functional sites across all Hsp70 proteins .', 'It appeared that the distributions of coevolutionary pMI scores and network centrality for functional sites were markedly shifted towards higher values of these parameters ( Fig 7C and 7D ) .', 'The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites .', 'We argue that coevolutionary and networking signatures of functional regions may be in harmony , acting as synchronizing forces that shape up the efficiency and robustness of allosteric regulatory mechanisms .', 'These conclusions echoed recent revelations that coevolutionary relationships may be intimately linked with protein dynamics and determine conformational heterogeneity and functional landscapes of protein structures [114–117] .', 'While many functional DnaK sites corresponded to high pMI residues , regulatory positions in the Sse1 structures featured markedly lower pMI values ( S4 Fig ) .', 'Hence , structural environment of functional sites in the Sse1 structures may be deficient in highly coevolving residues .', 'Moreover , the high centrality peaks in the Sse1 structures were less pronounced and not necessarily coincided with functional sites ( S4 Fig ) .', 'Since properly positioned coevolving residues may provide a primary vehicle for executing cooperative structural changes , the lack of allosteric communication in Sse1 [38–41] may be associated with dislocation of mediating centers and insufficient coevolutionary coupling between functional regions .', 'Notably , selection for specific functional constraints and lower substitution rates is a prominent feature of canonical Hsp70s such as DnaK [118] , whereas atypical Hsp70 chaperones , such as Sse1 , are characterized by the relaxed selection for functional constraints and higher substitution rates [119] .', 'Our results corroborated with this evidence by revealing the reduced dynamic and coevolutionary coupling between functional regions in the Sse1 structures , which may be linked with deficient allosteric signaling observed in this Hsp70 chaperone .', 'This may be contrasted with the observed convergence of dynamic and coevolutionary residue correlations in DnaK that may contribute to a highly cooperative allosteric mechanism with a broad network of mediating centers .', 'To conclude , our results suggested that the interplay between residue coevolution and protein dynamics may be important in shaping up the nature of allosteric regulatory mechanisms that could range from a population-shift mechanism in DnaK to an entropy-driven mechanism adopted by Sse1 .', 'To characterize topology and functional organization of allosteric interactions and communications in the Hsp70 structures , we performed a modular decomposition of protein structure networks using a community detection analysis [120] .', 'Ordinarily , protein structure modularity approaches are based on the residue contact matrix [121 , 122] and do not include dynamic or evolutionary information .', 'The network models of proteins based on residue contacts can often feature either the excessive modularity of rigid communities or produce overly flexible overlapping communities [123–126] .', 'However , an appropriate balance between structural rigidity and flexibility is a cornerstone of protein functions and adaptability [125 , 126] .', 'Using a community decomposition method , the residue interaction networks were divided into local modules in which residue nodes are strongly interconnected through both dynamic and coevolutionary correlations , whereas residues that belong to different communities may be sparsely connected and only weakly coupled .', 'We show that this model can adequately describe a balance between structural rigidity and flexibility within local communities that enables efficient inter-modular connectivity and promotes allosteric signaling in the Hsp70 structures .', 'An important question concerns functional significance of local communities and physical principles underlying modularity of the residue interaction networks .', 'To clarify these issues , we first analyzed the nature and composition of conserved communities that are shared between DnaK structures and can be preserved during allosteric structural changes .', 'An important finding was the emergence of conserved local communities that may be preserved to ensure structural stability and catalytic functions of the DnaK chaperone .', 'In both ADP-DnaK and ATP-Dnak structures , a shared stable community ( K70-E171-P143 ) was detected that links catalytic residues K70 , E171 with the allosteric switch P143 ( Table 1 , Fig 8 ) .', 'K70 and E171 are involved in catalysis of ATP hydrolysis , whereas P143 is a highly conserved residue that could act as a regulatory switch by assuming alternative conformations during ATP binding and hydrolysis [43] .', 'Another conserved community in the subdomain IA ( V142-F146-T154 ) protects structural stability of a critical regulatory residue F146 .', 'Mutation F146A can significantly reduce substrate release rate in the presence of ATP , thus pointing to the role of F146 in signal transmission from NB to SBD [53] .', 'The two conserved communities centered around rigid functional sites P143 and F146 may be necessary for coupling ATP binding to the inter-lobe movements during allosteric transitions [53] .', 'Several other conserved communities ( L324-F356-V331 ) and ( Q343-K270-M346 ) are shared by the DnaK structures forms and are responsible for structural stability of the subdomain IIA .', 'According to single molecule optical tweezer experiments , nucleotide binding in DnaK is dependent on thermal stability of the subdomain IIA [127] .', 'These studies also showed that stabilization and nucleotide-binding function of the lobe II in DnaK may be associated with structural preservation of residue cluster 330–345 in subdomain IIA .', 'Our results may rationalize these experiments by showing that structural and evolutionary preservation of local interacting modules ( L324-F356-V331 ) and ( Q343-K270-M346 ) in the subdomain IIA may protect stability and nucleotide binding function of the NBD core .', 'Intriguingly , the experime
['Introduction', 'Results and Discussion', 'Conclusions', 'Materials and Methods']
['Allosteric interactions in the Hsp70 proteins are linked with their regulatory mechanisms and cellular functions .', 'Despite significant progress in structural and functional characterization of the Hsp70 proteins fundamental questions concerning modularity of the allosteric interaction networks and hierarchy of signaling pathways in the Hsp70 chaperones remained largely unexplored and poorly understood .', 'In this work , we proposed an integrated computational strategy that combined atomistic and coarse-grained simulations with coevolutionary analysis and network modeling of the residue interactions .', 'A novel aspect of this work is the incorporation of dynamic residue correlations and coevolutionary residue dependencies in the construction of allosteric interaction networks and signaling pathways .', 'We found that functional sites involved in allosteric regulation of Hsp70 may be characterized by structural stability , proximity to global hinge centers and local structural environment that is enriched by highly coevolving flexible residues .', 'These specific characteristics may be necessary for regulation of allosteric structural transitions and could distinguish regulatory sites from nonfunctional conserved residues .', 'The observed confluence of dynamics correlations and coevolutionary residue couplings with global networking features may determine modular organization of allosteric interactions and dictate localization of key mediating sites .', 'Community analysis of the residue interaction networks revealed that concerted rearrangements of local interacting modules at the inter-domain interface may be responsible for global structural changes and a population shift in the DnaK chaperone .', 'The inter-domain communities in the Hsp70 structures harbor the majority of regulatory residues involved in allosteric signaling , suggesting that these sites could be integral to the network organization and coordination of structural changes .', 'Using a network-based formalism of allostery , we introduced a community-hopping model of allosteric communication .', 'Atomistic reconstruction of signaling pathways in the DnaK structures captured a direction-specific mechanism and molecular details of signal transmission that are fully consistent with the mutagenesis experiments .', 'The results of our study reconciled structural and functional experiments from a network-centric perspective by showing that global properties of the residue interaction networks and coevolutionary signatures may be linked with specificity and diversity of allosteric regulation mechanisms .']
['The diversity of allosteric mechanisms in the Hsp70 proteins could range from modulation of the inter-domain interactions and conformational dynamics to fine-tuning of the Hsp70 interactions with co-chaperones .', 'The goal of this study is to present a systematic computational analysis of the dynamic and evolutionary factors underlying allosteric structural transformations of the Hsp70 proteins .', 'We investigated the relationship between functional dynamics , residue coevolution , and network organization of residue interactions in the Hsp70 proteins .', 'The results of this study revealed that conformational dynamics of the Hsp70 proteins may be linked with coevolutionary propensities and mutual information dependencies of the protein residues .', 'Modularity and connectivity of allosteric interactions in the Hsp70 chaperones are coordinated by stable functional sites that feature unique coevolutionary signatures and high network centrality .', 'The emergence of the inter-domain communities that are coordinated by functional centers and include highly coevolving residues could facilitate structural transitions through cooperative reorganization of the local interacting modules .', 'We determined that the differences in the modularity of the residue interactions and organization of coevolutionary networks in DnaK may be associated with variations in their allosteric mechanisms .', 'The network signatures of the DnaK structures are characteristic of a population-shift allostery that allows for coordinated structural rearrangements of local communities .', 'A dislocation of mediating centers and insufficient coevolutionary coupling between functional regions may render a reduced cooperativity and promote a limited entropy-driven allostery in the Sse1 chaperone that occurs without structural changes .', 'The results of this study showed that a network-centric framework and a community-hopping model of allosteric communication pathways may provide novel insights into molecular and evolutionary principles of allosteric regulation in the Hsp70 proteins .']
['allosteric regulation', 'crystal structure', 'protein interaction networks', 'condensed matter physics', 'enzymology', 'network analysis', 'chaperone proteins', 'protein structure', 'dna', 'dna structure', 'crystallography', 'enzyme chemistry', 'computer and information sciences', 'solid state physics', 'proteins', 'enzyme regulation', 'proteomics', 'molecular biology', 'centrality', 'physics', 'biochemistry', 'nucleic acids', 'genetics', 'biology and life sciences', 'physical sciences', 'macromolecular structure analysis']