Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications
[['Allosteric regulation is an effective mechanism to control activity and dynamic adaptability of proteins in the cell during signal transduction , metabolism , catalysis , and gene regulation [1]–[4] .', 'Protein allostery is determined by the underlying thermodynamics of a system , where long-range interactions may be determined not only by large structural changes , reflected in the enthalpy contribution , but also by entropy-driven dynamic fluctuations around the average structure [5]–[8] .', 'The statistical model of allosteric regulation is based on the energy landscape theory of protein structure and dynamics , according to which the ensemble of preexisting conformational states and communication pathways can be modulated by allosteric perturbations [9]–[12] .', 'In this model , structural or dynamic perturbation at the effector site could cooperatively shift the relative distribution of the global conformational ensemble and stabilize the allosteric state .', 'Thermodynamics-based approaches associated structural perturbations and allosteric conformational changes with the free energy variations in the conformational ensembles of preexisting states [13]–[15] .', 'Elastic network models of protein dynamics [16] , [17] and the normal mode analysis [18] , [19] were combined with the theory of signal propagation [20] in the development of statistical models of allosteric communication pathways in proteins [21] , [22] .', 'The modern statistical view of protein allostery was further expanded to studies of allosteric protein interactions in signaling pathways and disease states [23]–[25] , investigations of disordered proteins [26] , modeling of molecular networks [27] , and mechanisms of allosteric protein inhibition [28] .', 'Graph-based theoretical approaches can provide a convenient characterization of protein topologies and allow for network-based analyses of protein structure and dynamics .', 'Structure-based network models were successfully used in simulations of protein folding mechanisms [29]–[31] , analysis of allosteric communications in proteins [32] , [33] , and prediction of binding site residues and regulatory sites in protein networks [34]–[37] .', 'These studies suggested that protein structure topologies could produce small-world networks in which a high local connectivity of residue nodes could be balanced by a smaller number of long-range interactions , giving rise to a high degree of interaction cooperativity .', 'Protein structure networks are often described as weighted graphs and use common measures of node centrality [38] to characterize the local connectivity of a particular node ( the degree of a node ) and global indices of node connectivity ( closeness and betweenness ) .', 'A combination of molecular dynamics ( MD ) simulations and protein structure network analysis [39]–[44] can characterize topology of allosteric communications in complex molecular assembles by analyzing network centrality and identifying stable communities of interconnected residues .', 'Dynamic-based network approaches employed MD-based contact maps of residue cross-correlations to yield a more accurate description of the network topology and robust description of allosteric communication pathways in tRNA–protein complexes [45] , cysteinyl tRNA synthetase [46] , imidazole glycerol phosphate synthase [47] , [48] , thrombin [49] , and the M2 muscarinic receptor [50] .', 'The Hsp90 protein ( 90 kDa heat-shock proteins ) is the most abundant molecular chaperone , whose functional versatility and structural adaptability to a broad spectrum of cochaperones and protein clients are regulated by allosteric interactions [51]–[54] .', "Rapid and efficient transmission of long-range conformational changes plays a vital role in allosteric regulation and may determine the regulatory mechanisms and cellular functions of signaling cascades that are under chaperone's control .", 'The class of Hsp90 chaperones is highly conserved and present in the cytosol of bacteria and all eukaryotes including the eukaryotic cytosolic Hsp90 , the bacterial homologue HtpG in E . coli , and the endoplasmic reticulum homologue Grp94 .', 'The Hsp90 chaperones share a conserved organization of a homodimer in which an N-terminal domain ( Hsp90-NTD ) is responsible for ATP binding , a middle domain ( Hsp90-MD ) complements the nucleotide binding site and binds client proteins , and a C-terminal domain ( Hsp90-CTD ) is involved in dimerization [51]–[54] .', 'Structural studies of Hsp90 in the unbound form and nucleotide-bound complexes from yeast [55] , E . coli HtpG [56]–[60] , and Grp94 homologue [61] determined that Hsp90 could assume a variety of distinct structural forms associated with the ATP binding and hydrolysis ( Figure 1 ) .', 'Structural studies supported a mechanism of conformational coupling to the ATPase cycle that is conserved among different species [60] and involves a coordinated transient dimerization of the Hsp90-NTDs and association of the Hsp90-NTD and the Hsp90-MD in the ATP-bound state , but not in the ADP-bound or apo states .', 'Recent biophysical studies using hydrogen exchange mass spectrometry ( HX-MS ) , electron microscopy ( EM ) , small-angle X-ray scattering ( SAXS ) , and single molecule fluorescence resonance energy transfer ( FRET ) assays confirmed that in the absence of cochaperones and substrate proteins Hsp90 operates in a stochastic conformational cycle associated with ATP binding and hydrolysis [62]–[67] .', 'Remarkably , conformational changes of the Hsp90 chaperone are only weakly coupled to nucleotide binding [64] and the Hsp90-ATPase functional cycle could be primarily determined by thermal fluctuations and spontaneous transitions between different functional forms [65] .', 'A comprehensive reconstitution of the Hsp90 functional cycle [66] , [67] demonstrated a controlled progression through distinct intermediate states that can be modulated by conformation-sensitive cochaperones .', 'The diverse regulatory mechanisms of the Hsp90 machinery are enabled by interactions with a large cohort of cochaperones that can tailor structural and functional plasticity of Hsp90 for demanding needs of client proteins .', 'Allosteric regulation of protein clients by the Hsp90 machine underlies its fundamental role in signal transduction networks associated with protein synthesis , assembly and activation of key signaling proteins driving tumor development and progression .', 'An intriguing aspect of cochaperone function is that cytosolic Hsp90 is the only form of Hsp90 that requires cochaperones for function .', 'The recent structural and functional investigations revealed a diversity of mechanisms by which cochaperones may assist Hsp90 in modulating the progression of the ATPase cycle and recruiting protein clients [68]–[70] .', 'The analysis of cochaperone integration in the ATPase cycle confirmed that the Hsp90 binding with cycle-accelerating cochaperones ( Aha1 , Cpr6 ) and cycle-inhibiting cochaperones ( p23 ) can modulate the progression of the conformational transitions by switching the stochastic equilibrium into a more deterministic succession of specific chaperone states [71] .', 'A dynamically controlled binding and release of cochaperones from Hsp90 could modulate the thermodynamics of the ATPase cycle and control conformational transitions required for proper loading and release of the substrate proteins [72]–[74] .', 'In recent years computational studies from several groups have employed structural and dynamic approaches to quantify mechanistic aspects of Hsp90 regulation .', 'Our early studies provided the first atomic resolution models of allosteric regulation in the Hsp90 chaperone and presented evidence of a cross-talk between the N- and C-terminal binding sites of Hsp90 [75] , [76] .', 'These computational predictions were subsequently confirmed in the fluorescence-based single molecule assay [63] revealing a strong influence of the ATP binding and hydrolysis in the nucleotide site on the dynamics of C-terminal dimerization .', 'Integration of computational and experimental approaches subsequently led to structural characterization of the binding sites and prediction of allosteric inhibitors targeting the C-terminal binding site in Hsp90 [77]–[80] .', 'A combination of atomistic and coarse-grained approaches with the energy landscape was used to systematically investigate functional dynamics and global motions of the Hsp90 chaperone [81] .', 'This approach identified a network of conserved regions that may be utilized by the molecular chaperone for regulation of the inter-domain communications and control of ATP hydrolysis .', 'All-atom simulations of the Hsp90 crystal structures from different species confirmed that functional dynamics of Hsp90 may be determined by the motion of quasi-rigid domains involving a set of conserved functional residues from the inter-domain hinge sites [82] .', 'Force-distribution analysis based on atomistic simulations of the Hsp90 structures detected allosteric communication pathways connecting the nucleotide-binding site to a distant hinge region and to a putative protein client binding site [83] .', 'Structural and thermodynamic analysis of conformational transitions in Hsp90 using MD simulations described large-scale structural rearrangements and characterized key interactions governing the conformational change [84] .', 'Allosteric regulation of the Hsp90 dynamics and stability by cochaperones and client proteins was pursued in our recent studies [85]–[87] .', 'Functional dynamics analysis of the Hsp90-cochaperone complexes revealed that allosteric interactions in Hsp90 can be selectively modulated by p23 and Aha1 cochaperones that could stabilize specific chaperone conformations [85] .', 'Moreover , the interaction networks in the Hsp90 complexes with p53 [86] and client recruiter cochaperones Cdc37 , Sgt1 and Rar1 [87] may produce small-world networks in which highly connected mediating residues at the intermolecular interfaces correspond to the functional hot spots .', 'In this work , we integrated atomistic simulations of the Hsp90 crystal structures with the structural stability analysis and protein structure network modeling to characterize organization and evolution of the interaction networks and allosteric communications during ATPase-coupled conformational changes of the chaperone .', 'The dynamics-based force constant analysis determines a network of evolutionary conserved and structurally stable residues that can function as mediating sites of allosteric communications in the Hsp90 proteins .', 'Mapping of the principal conformational space of chaperone motions with the network centrality parameters characterizes the dynamic evolution of the conserved interaction networks that can act synchronously with the shifts in the conformational ensemble and determine structural stability of functional states .', 'This analysis can reconcile a broad range of structural and functional experiments by revealing that functional hotspots of the chaperone activity could serve as global mediating centers of structural stability and allosteric communications .', 'We show that dynamic coupling between these functional residues may be important to maintain the robustness of the inter-domain allosteric interactions and modulate the network efficiency in response to functional requirements of the Hsp90-ATPase cycle .', 'The results of this study suggest that a small number of functional residues may be utilized by the chaperone machinery as central regulators of multiple functions , including structural stability , allosteric communications , and binding with cochaperones and client proteins .'], ['Multidomain protein structures have complex and variable residue distributions of structural rigidity and flexibility that have evolved to maintain robustness of biological systems to random perturbations and mutations , while allowing for adaptability to new functions .', 'MD simulations of the Hsp90 chaperone in different functional forms were combined with the structural stability analysis to characterize the organization and modularity of structurally rigid and conformationally flexible residues during conformational equilibrium changes .', 'The following specific objectives were addressed in simulations:', '( a ) to determine conserved features and principal differences in the conformational mobility and distribution of structurally stable regions in the Hsp90 chaperone states;', '( b ) to characterize nucleotide-specific changes in the Hsp90 dynamics during the ATPase cycle; and', '( c ) to describe functional coupling between structurally rigid and conformationally flexible regions that may be required for allosteric signaling in the Hsp90 system .', 'A comparative analysis of the conformational ensembles highlighted fundamental aspects of the Hsp90 dynamics that may be exploited by the chaperone to ensure proper progression of the ATPase cycle .', 'In agreement with the structural studies [56] , [57] , we found that both apo-HtpG and ADP-bound HtpG states were quite flexible and displayed large thermal fluctuations and the increased conformational mobility as evident from the root mean square fluctuation ( RMSF ) of the backbone residues and computed B-factors ( Figures 2A , B ) .', 'Although apo-HtpG adopts an open conformation in the crystal and SAXS structures [56] , the HtpG-NTDs could experience only local thermal motions and the HtpG-MDs remained closed ( Figures 2 , S1 ) .', 'Overall , the relative movements between the NTDs and MDs were relatively small in the apo-HtpG structure , which is consistent with the results of FRET experiments [66] .', 'A significantly greater mobility was detected in all domains of the ADP-bound HtpG complex , particularly in the regulatory lid region of the NTD ( Figure 2B , S1 ) .', 'Moreover , conformational changes in the ADP-HtpG complex were not limited to the HtpG-NTDs , but also revealed larger protein movements and a partial separation of the HtpG-MDs ( Figure 3 A , B ) .', 'A fairly large conformational change observed in simulations of ADP-HtpG was primarily driven by global motions near the NTD-MD hinge region .', 'In contrast , thermal fluctuations of the ATP-bound HtpG were considerably smaller and the chaperone remained in a closed conformational form throughout the simulation period ( Figure 3C , D ) .', 'In the ADP-HtpG structure , the nucleotide-specific protection of amide hydrogens was experimentally observed only in the binding site of HtpG-NTD ( residues 90–98 ) , whereas some deprotection was widely spread across multiple domains [62] .', 'Our results are consistent with these HX-MS experiments , as high flexibility was observed in the nucleotide-free and ADP-bound HtpG forms , while the addition of ATP could lead to a considerable reduction of thermal motions and stabilization of the closed functional state ( Figures 2 , 3 ) .', 'Computational predictions are also in an agreement with the atomistic simulations and force distribution analysis of the HtpG chaperone that revealed similar movements of the NTDs and MDs [83] .', 'Noteworthy , the time scale of current MD simulations may not allow to observe a complete conformational equilibrium between well-defined opened and closed ADP-HtpG states , as the equilibrium transitions between these states could be extremely slow [66] .', 'Nonetheless , in agreement with the experiments , simulations showed that the ADP and ATP nucleotide binding can differentially modulate equilibrium between open and closed dimer conformations of HtpG .', 'Moreover , the observed conformational flexibility and weakened inter-domain interactions in the ADP-HtpG chaperone are consistent with the fluorescence spectroscopy [57] and HX-MS dynamic studies of HtpG [62] , in which the ADP-bound HtpG form could not be detected as a stable intermediate in solution and was proposed to exist only transiently .', 'MD simulations of the ATP-bound yeast Hsp90 demonstrated small thermal fluctuations occurring near the crystallographic conformation , where the NTDs and CTDs remained in a closed structural arrangement ( Figure 2C ) .', 'These results are consistent with the experimental data and our previous studies [76] , [81] confirming that structural stability of the inter-domain interactions is a fundamental characteristic of the ATP-bound Hsp90 complex .', 'A different distribution of structurally stable regions was observed in simulations of the ATP-bound Grp94 homologue ( Figure 2D ) .', 'While the Grp94-NTDs appeared to be mobile , the thermal movements of the MD and CTD regions were fairly small , which is consistent with the experimental data , since nucleotide binding had a small effect on conformational rearrangements in the Grp94 chaperone , yet even a partial truncation of the peripheral CTD regions may lead to a considerable loss of the hydrolysis activity [61] .', 'The results of MD simulations were utilized in the force constant analysis of structural stability performed for different functional forms of Hsp90 [88] , [89] .', 'In this approach , the fluctuations of the mean distance between each residue and the rest of the protein are translated into force constants that measure the energy cost of the residue displacement during equilibrium simulations .', 'The residues with high force constants are typically structurally rigid and abrupt changes between maxima and minima in the distributions may signal the emergence of boundaries between structurally rigid modules and flexible regions , often pointing to the inter-domain hinge sites .', 'The hypothesis tested in this analysis is that functional hotspots of the chaperone activity could be involved in mediating structural stability and allosteric communications in Hsp90 by bridging high and low stability regions near the inter-domain hinge sites .', 'In this formulation , structural stability of the inter-domain regions may be an important determinant that controls global chaperone movements and ensures normal progression of the ATPase cycle .', 'The residue-based force constant profiles can be also associated with allosteric communication capabilities .', 'We previously formulated a model that defined communication propensities of protein residues by relating the mean square fluctuations between a pair of residues to their commute time [85] .', 'According to this model , structurally stable residues with high force constants may display small fluctuations in their distances to other residues and often correspond to effectively communicating rigid sites .', 'We analyzed the organization of structurally stable residue modules in the Hsp90 structures to identify and characterize key functionally residues that may contribute to structural stability , allosteric communications , and chaperone activity .', 'The peaks of the force constant distributions were also mapped onto conformational mobility profiles in the essential space of low frequency motions in order to determine if structurally stable regions can act synchronously with the shifts in the conformational ensemble .', 'The force constant profiles for the HtpG structures were characterized by a moderate number of high peaks and mostly reflected conformationally flexible nature of the apo ( Figure 4 ) and ADP-bound HtpG states ( Figure 5 ) .', 'Moreover , the distribution peaks were narrow and often corresponded to single rigid residues rather than consolidated structurally rigid modules .', 'The NTD-MD hinge site in HtpG is formed by conserved residues 332-LNVSREIL-339 that include catalytic R336 , while the MD-CTD hinge region consists of residues 378-FGLVLKE-384 and 393-QEAIAKL-399 ( Figure 1 ) .', 'Importantly , the identified structurally stable regions mapped onto a number of well-characterized functional residues of the HtpG homologue , including hinge sites at the inter-domain regions ( Figure 4B , 5B ) .', 'Notably , the force constant peaks for apo-HtpG ( Figure 4 ) and ADP-HtpG ( Figure 5 ) corresponded to functionally critical H446 and L447 residues that contribute to stabilization of the MD-CTD interfacial regions .', 'SAXS experiments have identified that pH-dependent conformational equilibrium for apo-HtpG that can be effectively modulated by mutations of a single residue H446 [58] .', 'According to this study , the H446K mutant can shift conformational preferences towards a more compact Grp94-like state , which is adopted by HtpG-WT at low pH , whereas H446E mutation can push the equilibrium to a more extended conformation that is assumed by HtpG-WT at high pH . The force constant profiles of apo-HtpG and ADP-HtpG also exhibited peaks in the functionally important region 465-DEWMMN-470 ( Figures 4 , 5 ) .', 'This motif bridges the HtpG-MD and HtpG-CTD regions and contains functionally important residues E466 , W467 , and N470 whose mutants could significantly compromise the ATPase activity and diminish the ability of the chaperone to activate a diverse range of substrate proteins [74] .', 'Another common structurally rigid site 535-TPAIV-539 is situated in the HtpG-CTD and contains a conserved regulatory switch I538 [90] .', 'Hence , we found that functional chaperone residues could often correspond to the force constant profile peaks and occupy the inter-domain regions bordering high and low stability regions .', 'The force constant profiling of the ATP-bound yeast Hsp90 suggested a considerable stabilization of the NTD and MD residues , especially the inter-domain interfaces , as evident from the reduced number of conformationally flexible ( low force constant ) residues in these regions ( Figure 6 ) .', 'Importantly , this profile revealed the emergence of broad peaks , reflecting consolidation of structurally stable residues into interconnected rigid modules .', 'The force constant peaks in the Hsp90-NTDs corresponded to functional residues T22 , Y24 , and A41 that contribute to stabilization of the inter-monomer interface and determine a closed structural arrangement of the ATP-Hsp90 dimer [91] , [92] .', 'Mutations of the Hsp90-NTD residues ( T22 , Y24 , and T101 ) could modulate the ATPase activity and reduce client protein activation .', 'Interestingly , a single mutation T101I could disfavor the ATP-induced conformational switch in Hsp90 and reinforce interactions with the Cdc37 cochaperone , whereas T22I can induce this conformational switch and has the opposite effect on binding with Cdc37 [92] , [93] .', 'Mutations of Y24 and T22 to glutamic acid can also markedly impair Hsp90-ATPase activity and the produced chaperone defect in cells could be even more severe [93] .', 'The broad peaks in the distribution also corresponded to structurally stable modules from the Hsp90-MD ( residues 348-VFITDE-353 , 376-LNLSREML-383 , and 426-KLGV-429 ) .', 'These interaction clusters include functionally important residues near the NTD-MD interface region ( V348 , F349 , I350 ) that are involved in stabilization of the inter-domain interactions required for the formation of the active dimer .', 'Structural and functional analysis of yeast Hsp90 has demonstrated that F349A and F349Q mutations could severely reduce ATPase activity in vitro and impair client activation function [94] .', 'Another important peak in the force constant distribution corresponded to the functional loop 376-LNLSREML-383 that harbors the critical catalytic residue Arg-380 and hydrophobic residues L376 , L378 ( Figure 6C ) .', 'These functional residues act synergistically using a combination of the charged interactions and cross-monomer hydrophobic interactions to stabilize the closed and catalytically active dimer conformation [95] .', 'A sharp peak in the force constant profile was also associated with the stable motif 426-KLGV-429 near the MD-CTD inter-domain hinge ( Figure 6 ) .', 'This motif belongs to the helix 2 ( residues 412–431 ) of the three-helix bundle ( Figure 1 ) that displayed the increased protection in HX-MS experiments [62] .', 'Smaller peaks were also observed for functional residues ( E507 , Y508 , and T511 ) whose mutations can reduce chaperone activity in vivo and adversely affect binding of client proteins [74] .', 'These findings reinforced the importance of the inter-domain hinge regions as stable functional modules that can control conformational transitions during the Hsp90-ATPase cycle .', 'Structural studies of the Grp94 homologue in complexes with AMP-PNP and ADP [61] have revealed a partially closed conformation that is catalytically silent and conformationally insensitive to the identity of the bound nucleotide .', 'The rigidity profile of the ATP-bound Grp94 ( Figure S2 ) revealed low force constant values ( high conformational flexibility ) for the NTD residues and the NTD-MD region of the second monomer .', 'The weakened inter-domain interactions in ATP-Grp94 may be responsible for the lack of productive dimerization interactions between the two NTDs and explain why ligand-induced conformational changes in Grp94 are typically limited only to the N-termini regions [61] .', 'We also observed that the Grp94-NTD residues near the inter-domain boundaries may exhibit a different level of flexibility in two monomers .', 'At the same time , high force constant values were seen for residues located near the MD-CTD interface .', 'The force constant peaks in the Hsp90-MD corresponded to mostly hydrophobic residue clusters 494-LGVIED-499 , 425-MMPKYL-430 , and 658-MERIM-662 ( Figure S2 ) .', 'Consistent with these results , structural experiments indicated that a conserved M658-M662 pair could present a client-binding site in Grp94 [61] .', 'Conformational flexibility of the Grp94-NTDs may partly explain why functional residues , which are located at the NTD-MD interface , could become immune to mutations .', 'Indeed , mutations of the Grp94 residues R448 ( corresponding to R380 in yeast ) , Q452 ( Q384 in yeast ) and hydrophobic patch 416-VF-417 ( 348-VF-349 residues in yeast Hsp90 ) do not induce loss of ATPase activity [96] , [97] .', 'In contrast , some of these residues in yeast ATP-Hsp90 ( R380 , 348-VF-349 ) can act as ATP-sensitive stabilizers of the inter-domain interactions and dimerization [98] , [99] .', 'Our analysis captured these important differences between yeast Hsp90 and Grp94 , as the corresponding residues exhibited high force constants ( high structural stability ) in yeast Hsp90 ( Figure 6 ) , but displayed moderated force constant values and conformational flexibility in Grp94 ( Figure S2 ) .', 'In summary , the force constant analysis of structural stability recapitulated a broad range of experimental observations by linking structural stability and communication propensity of protein residues with their importance in the ATPase cycle and chaperone activity .', 'We compared our computational results with the HX-MS and fluorescence spectroscopy analysis of the conformational dynamics of HtpG and yeast Hsp90 in solution [62] .', 'In these experiments , the addition of ADP induced a small but broadly distributed amide hydrogen deprotection and a greater flexibility in the HtpG-NTD lid segment ( residues 108–119 ) , the HtpG-MD ( residues 279–299 and 401–416 ) and most of the HtpG-CTD regions .', 'Consistent with these experiments , our analysis revealed that force constant values for these residues were reduced in the ADP-HtpG and corresponded to the distribution minima , thus pointing to the conformationally flexible regions ( Figure 5A ) .', 'A considerably stronger effect was experimentally observed in the ATP-HtpG complex , where amide hydrogen protection and the increased structural rigidity was detected in the HtpG-NTD ( residues 2–19 , 21–31 , 90–98 , 121–127 , 192–206 ) and HtpG-MD regions ( residues 319–334 , and 336–359 ) [62] .', 'Experimental measurements are typically confined to the backbone amide protons , which are involved in hydrogen bonding for the maintenance of secondary structure , yet amides buried from the solvent but not hydrogen bonded may also have very slow exchange rates .', 'The experimentally observed nucleotide-dependent changes in the hydrogen–deuterium exchange for the Hsp90 structures may thus depend on both structural rigidity and solvent accessibility .', 'To facilitate a quantitative comparison with the experimental HX-MS data , we supplemented the force constant stability predictions with the ensemble-based analysis of solvent accessibility .', 'In order to differentiate between buried residues located near the protein surface and inside the protein core , we used a computational procedure for calculating the depth of a residue from the protein surface [100] .', 'A differential plot of the residue depth profiles was computed for apo-HtpG and ATP-HtpG using MD-based equilibrium ensembles ( Figure S3A ) .', 'Consistent with the HX-MS data , this analysis not only detected the increased and broadly distributed protection from solvent in the ATP-HtpG structure , but also closely mapped these changes onto the respective NTD ( residues 90–96 , 121–127 ) and MD regions ( residues 319–359 ) .', 'Nucleotide-induced protection in the ATP-bound HtpG reflected a shift in the conformational equilibrium towards a closed conformational form that was observed in simulations ( Figure 3C , D ) .', 'Our analysis also indicated that the improved protection of the NTD-MD hinge site ( residues 332-LNVSREIL-339 ) may be important in modulating allosteric communications in the closed conformation of the ATP-HtpG complex .', 'In agreement with the experiments , these results indicated that conformational equilibrium changes in HtpG may be strongly coupled to the nucleotide turnover by following a mechanical ratchet mechanism [66] .', 'In this mechanism , the HtpG chaperone can experience opening and closure of the HtpG-NTDs over a large distance via thermal fluctuations , whereas ATP binding can selectively stabilize a closed conformational form of the chaperone .', 'In yeast Hsp90 , the effect of ATP-induced protection was especially pronounced in the nucleotide-bound lid segment , while small protection was broadly distributed in the Hsp90-MDs and Hsp90-CTDs .', 'The hydrogen exchange profile in the yeast ATP-Hsp90 pointed to the increased rigidity in the nucleotide-interacting lid residues 91–105 and 118–124 , the Hsp90-MD ( residues 341–349 , 418–435 , and 477–492 ) and in the Hsp90-CTD residues 622–642 [62] .', 'The force constant analysis of ATP-Hsp90 predicted structural stabilization in these regions , particularly emphasizing a critical role of the NTD lid motif and the inter-domain MD residues forming modules of strongly interacting residues ( Figure 6 ) .', 'To further quantify a comparison with the HX-MS data , we also computed the ensemble-averaged residue depth profiles for apo and ATP-bound yeast Hsp90 ( Figure S2B ) .', 'This analysis reproduced a moderate but broadly distributed effect of the nucleotide binding .', 'Consistent with the HX-MS data , the increased amide protection was properly mapped onto the lid segment ( residues 91–105 and 118–124 ) , the Hsp90-MD regions ( residues 380–435 , 470–492 ) and the Hsp90-CTD segments ( residues 540–600 ) ( Figure S3B ) .', 'The increased protection was also detected for the hinge regions ( residues 376-LNLSREML-383 , 426-KLGVHE-431 ) .', 'Computational predictions using both the force constant analysis and the residue depth profiling pointed to the ATP-induced stabilization of the inter-domain regions .', 'Noteworthy , small deprotection peaks corresponded to single flexible residues distributed across all domains , while a consistent pattern of the enhanced protection in functional regions may be statistically significant and reflect the effect of ATP-induced stabilization of a closed dimerized state .', 'Our results are consistent with the experimental findings suggesting that conformational changes in yeast Hsp90 are less sensitive to the nucleotide binding and may be driven by slow thermal fluctuations between the open and closed states [66] .', 'The recruitment of cochaperones into Hsp90 can turn the stochastic conformational changes in yeast Hsp90 into a successive progression of functional states by precisely tuning structural stability of the nucleotide-bound complexes during the ATPase cycle [71] .', 'We also employed a structure-based network analysis of the Hsp90 chaperones to characterize the organization of local hubs and stable interaction communities as a simple and robust metric for evaluation of structural stability .', 'Protein structure networks were constructed by incorporating bo
['Introduction', 'Results/Discussion', 'Materials and Methods']
['A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks .', 'In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones .', 'We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble .', 'The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle .', 'Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication .', 'Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues .', 'The network analysis has revealed a relationship between structural stability , global centrality and functional significance of hotspot residues involved in chaperone regulation .', 'We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network .', 'The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes .', 'This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks .']
['Functional versatility and structural adaptability of the Hsp90 chaperones are regulated by allosteric interactions that allow for diverse functions including modulation of ATP hydrolysis and binding with cochaperones and client proteins .', 'By integrating molecular simulations and network-based approaches we have characterized conformational dynamics and allosteric interactions in different functional forms of Hsp90 .', 'The network centrality analysis and structural mapping of allosteric communications have revealed a small-world organization of the interaction network that is mediated by functionally important residues of the Hsp90 activity .', 'We have found that effective allosteric communications in the Hsp90 chaperone may be provided by structurally stable residues that exhibit high centrality properties .', 'Nucleotide-specific rewiring of the network topology and assortative organization of functional residues may protect the active form of the chaperone from random perturbations and detrimental mutations .', 'These results have confirmed that allosteric interactions in the Hsp90 chaperone may be determined by a small-world network of functional residues that can regulate the network efficiency and resiliency by modulating the statistical ensemble of communication pathways in response to functional requirements of the ATPase cycle .']
['biomacromolecule-ligand interactions', 'molecular complexes', 'molecular dynamics', 'classical mechanics', 'molecular mechanics', "newton's laws of motion", 'protein folding', 'protein structure', 'thermodynamics', 'physical chemistry', 'computer and information sciences', 'proteins', 'biophysics theory', 'chemistry', 'biophysics', 'molecular biology', 'motion', 'macromolecular complex analysis', 'physics', 'biochemistry', 'computer modeling', 'biology and life sciences', 'physical sciences', 'computational chemistry', 'computational biology', 'macromolecular structure analysis', 'biophysical simulations']