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Phosphorylation ofEscherichia coliCheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY.FliMNwith and without D13K-Y106W have shown FliMNbound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMNand D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D13K at the D57 phosphorylation site and Y/W106 at the FliMNinterface; namely the closure of the α4-β4 hinge and inward rotation of Y/W106 with W58. We used hydroxy-radical foot-printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other sidechains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay sidechains reported by XFMS confirmed the intermediate conformation of the native CheY.FliMNcomplex, the inactive state of free D13K-Y106W CheY and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch with Y/W106 sidechain burial correlated with increased FliMNaffinity. Activation entrained CheY fold stabilization to FliMNaffinity. The CheY network could be partitioned into four dynamically coordinated community sectors. Residue substitutions mapped to sectors around D57 or the FliMNinterface according to phenotype. FliMNincreased sector size and interactions. These sectors fused between the substituted K13K-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far.Statement of SignificanceCheY affinity for FliMN, its binding target at the flagellar motor, is increased by phosphorylation to switch rotation sense. Atomistic simulations based on CheY and CheY.FliMNcrystal structures with and without the phospho-mimetic double substitution (D13K-Y106W) showed CheY compaction is entrained to increased FliMNaffinity. Burial of exposed aromatic sidechains drove compaction, as validated by tracking sidechain solvent accessibility with hydroxyl-radical foot-printing. The substitutions were localized at the phosphorylation pocket (D13K) and FliMNinterface (Y106W). Mutual information measures revealed these locations were allosterically coupled by a specialized conduit when the conformational landscape of FliMN-tethered CheY was modified by the substitutions. Novel surfaces stabilized by the conduit may bind additional motor sites, essential for the high cooperativity of the flagellar switch.

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modelling and simulations in conjunction with biochemical characterisation to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor-substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering. Federal Scholarship Board/Presidential Special Scholarship Scheme for Innovation and Development (PRESSID), Nigeria; Sichuan Science and Technology Program

Motivation A deleterious amino acid change in a protein can be compensated by a second-site rescue mutation. These compensatory mechanisms can be mimicked by drugs. In particular, the location of rescue mutations can be used to identify protein regions that can be targeted by small molecules to reactivate a damaged mutant. Results We present the first general computational method to detect rescue sites. By mimicking the effect of mutations through the application of forces, the double force scanning (DFS) method identifies the second-site residues that make the protein structure most resilient to the effect of pathogenic mutations. We tested DFS predictions against two datasets containing experimentally validated and putative evolutionary-related rescue sites. A remarkably good agreement was found between predictions and experimental data. Indeed, almost half of the rescue sites in p53 was correctly predicted by DFS, with 65% of remaining sites in contact with DFS predictions. Similar results were found for other proteins in the evolutionary dataset. Availability and implementation The DFS code is available under GPL at https://fornililab.github.io/dfs/ Supplementary information Supplementary data are available at Bioinformatics online.

The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects., Author summary Protein kinases play central roles in intracellular signalling. Auto-phosphorylation by bound nucleotide typically precedes phosphate transfer to multiple substrates. Protein conformational changes are central to kinase function, altering binding affinities to change cellular location and shunt from one signal pathway to another. In the brain, the multi-subunit kinase, CaMKII is activated by calcium-calmodulin upon calcium jumps produced by synaptic stimulation. Auto-transphosphorylation of a regulatory peptide enables the kinase to remain activated and mediate long-term behavioural effects after return to basal calcium levels. A database of mutated residues responsible for these effects is difficult to reconcile solely with impaired nucleotide or substrate binding. Therefore, we have computationally generated interaction networks to map the conformational plasticity of the kinase domains where most mutations localize. The network generated from the atomic structure of a phosphorylated dimer resolves protein sectors based on their collective motions. The sectors link nucleotide and substrate binding sites in self-reinforcing relays between regulatory peptides. The self-reinforcement is strengthened by phosphorylation consistent with the reported positive cooperativity of kinase activity with calcium-calmodulin concentration. The network gives a better match with the mutations and, in addition, reveals target sites for drug development.

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses., Author summary A low oxygen condition, called hypoxia, often occurs in tumor masses and generally correlates with worse prognosis. Cells in a tumor react to low oxygen levels with a metabolism modification induced by the activation of hypoxia inducible factors (HIFs) through dimerization with a partner protein and binding to a DNA target. Disrupting this protein-protein interaction could be a potential therapeutic strategy, but directly interfering with dimer formation can be troublesome because of the difficulty to design drugs that bind to protein interfaces. However, ligands that bind internal protein cavities can indirectly perturb the interfaces reducing dimers stability. Albeit protein crystallography had offered a detailed static picture of a HIF dimer bound to candidate inhibitors, it is not able to describe either the perturbation caused by binding or the molecular mechanism of dimer destabilization. Here we exploit molecular dynamics to identify the crucial interfaces in the HIF dimer stabilization and, by comparing the results obtained in the bound and unbound forms, we reveal the mechanism of ligand inhibition at atomic detail. All these findings will guide toward the design of improved dimerization inhibitors, to contrast cell survival in tumor masses.

The proton-driven ATP synthase (FoF1) is comprised of two rotary, stepping motors (Fo and F1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other FO subunits (ab2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring X-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring which varies between organisms. Molecular determinants were identified by covariance analysis of residue co-evolution and a structural alphabet based local dynamics correlations. The residue co-evolution gave a read-out of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the mid-membrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intra-subunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Non-contact couplings between IB-GGGG motifs matched the co-evolution signal as well as contact couplings. The residue co-evolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.

Molecular dynamics simulations examined how single transphosphorylation between adjacent subunits of calcium calmodulin dependent protein kinase II (CaMKII) effects conformational coupling. One (receiver) kinase captures and phosphorylates the regulatory domain of a donor kinase forming a chained dimer. Identified substrate binding sequences formed nodes in long-range, parallel signal relays linking regulatory peptides in adjacent subunits. Monomer subunit architecture was co-opted in the auto-phosphorylating dimer but the community hub shifted from the nucleotide binding pocket to a hydrophobic cluster between the captured peptide and a key helix in its binding pocket. Phosphorylation of this peptide modulated the dielectric of the pocket to reduce helix flexibility and collective motions of the receiver kinase domain. The signal relay was strengthened with phosphorylationdependent conformational transitions completed within hundred nanoseconds. The network couplings should guide allosteric inhibitor design. Chained dimers, in addition to intra-holoenzyme spread, may facilitate exchange between holoenzymes to prolong activated population lifetimes.

IntroductionConformational changes associated with protein function often occur at timescales inaccessible to unbiased Molecular Dynamics (MD) simulations, consequently different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in pre-calculated libraries known as Structural Alphabets (SA)[1], can be used to explore the landscape of global protein conformations in MD simulations.MethodsSAs were successfully used to analyze protein dynamics after simulation[2,3]. Here we define a novel SA-based Collective Variable (CVSA) to bias the sampling of backbone conformations of protein fragments towards experimentally preferred local states[4].ResultsWe find that: a) Enhancing the sampling of native local states allows recovery of global folded states, both in Metadynamics and in Steered MD, when the local states are encoded by strings of SA letters derived from the native structures. b) Global folded states are still recovered when the information on the native local states is reduced by using a low-resolution SA, where the original letters are clustered into macrostates. The macrostates provide the approximate shape of the fragments, while sampling with the atomistic force field allows the structure to adopt the native conformation of the specific amino acid sequence. c) SA strings derived from collections of structural motifs can be effectively used to sample alternative conformations of pre-selected regions. These findings have potential impact on a wide range of applications, from protein model refinement to folding and design.References1. Pandini A., Fornili A., Kleinjung J., BMC Bioinformatics 11:97 (2010).2. Pandini A., Fornili A., Fraternali F., Kleinjung J., FASEB J. 26:868 (2012).3. Pandini A., Fornili A., Fraternali F., Kleinjung J., Bioinformatics 29:2053 (2013).4. Pandini A., Fornili A., submitted.

Background Molecular dynamics (MD) simulations are powerful tools to investigate the conformational dynamics of proteins that is often a critical element of their function. Identification of functionally relevant conformations is generally done clustering the large ensemble of structures that are generated. Recently, Self-Organising Maps (SOMs) were reported performing more accurately and providing more consistent results than traditional clustering algorithms in various data mining problems. We present a novel strategy to analyse and compare conformational ensembles of protein domains using a two-level approach that combines SOMs and hierarchical clustering. Results The conformational dynamics of the α-spectrin SH3 protein domain and six single mutants were analysed by MD simulations. The Cα's Cartesian coordinates of conformations sampled in the essential space were used as input data vectors for SOM training, then complete linkage clustering was performed on the SOM prototype vectors. A specific protocol to optimize a SOM for structural ensembles was proposed: the optimal SOM was selected by means of a Taguchi experimental design plan applied to different data sets, and the optimal sampling rate of the MD trajectory was selected. The proposed two-level approach was applied to single trajectories of the SH3 domain independently as well as to groups of them at the same time. The results demonstrated the potential of this approach in the analysis of large ensembles of molecular structures: the possibility of producing a topological mapping of the conformational space in a simple 2D visualisation, as well as of effectively highlighting differences in the conformational dynamics directly related to biological functions. Conclusions The use of a two-level approach combining SOMs and hierarchical clustering for conformational analysis of structural ensembles of proteins was proposed. It can easily be extended to other study cases and to conformational ensembles from other sources.

Dynamical properties of proteins may have a significant role in regulating protein-protein interactions. In particular, intrinsic disorder and disorder-order transitions have been claimed to be especially involved in the binding of proteins known as “hubs” [1], which are characterized by a high level of connectivity in protein-protein interaction (PPI) networks. We therefore started to investigate the dynamical properties of hubs through molecular simulations, to assess the role of conformational flexibility in promoting “promiscuity” of interactions.For this study, a dataset of proteins with known structure and interaction partners was first prepared. To cope with the incompleteness of the interaction data contained in the Protein Data Bank (PDB), we mapped the PPI database IntAct [2], which collects interactions from a wide range of experimental techniques, onto the structure-based database PiSite [3]. The proteins were then partitioned into ‘classes’ with increasing number of interactions.A preliminary survey of the dynamical properties of each class was done using two independent approaches, namely tCONCOORD [4] and the Gaussian Network Model [5]. For each complex, the interfaces were extracted using the POPSComp method [6,7]. The availability of information on both the dynamics and the interaction properties will allow to determine possible correlations between flexibility and binding diversity.[1] Dosztnyi et al., J. Proteome Res., 5, 2985-2995 (2006).[2] Kerrien et al., Nucleic Acids Res., 35, D561-D565 (2006).[3] Higurashi et al., Nucleic Acids Res., 37, D360-D364 (2009).[4] Seeliger et al., Structure, 15, 1482-1492 (2007).[5] Bahar et al., Folding & Design, 2, 173-181 (1997).[6] Fraternali et al., Nucleic Acids Res., 30, 2950-2960 (2002).[7] Kleinjung et al., Nucleic Acids Res., 33, W342-W346 (2005).

IntroductionAllosteric inhibitors were shown to be effective in targeting multiple conformational states and more selective than competitive inhibitors [1]. However, allosteric signal transmission is difficult to model because often mediated by small local changes propagating along multiple pathways in the protein structure [2]. Therefore, we developed a method to identify these pathways by an information-theoretic analysis of Molecular Dynamics (MD) simulations.MethodsFirst, MD ensembles are mapped onto a canonical set of representative fragments, i.e. a set of discrete 4-residue states [3]. By this mapping, the contribution of rigid fragment roto-translations is removed and relevant changes are detected, even if subtle. Second, local motions are modelled as transitions between the fragment states. This removes background noise from harmonic high-frequency fluctuations. Third, the communication between parts of the molecule is modelled by the coupling of fragment transitions. These are resolved spatially and temporally. The time-averaged spatial couplings are analyzed with a network model of fragment correlations. Then communication pathways are characterised by time-resolved analysis of fragment couplings.ResultsThe method was applied to the NtrC, CheY, and FixJ receiver domains. The results are consistent with the emerging picture of allosteric regulation [2]: a pre-organized network of fragment couplings connects the allosteric and functional sites already in the inactive state, and fragments with high network centrality (hubs) are part of functionally relevant regions. Critical nodes on the signalling pathways correspond to residues experimentally known to affect the kinetics and thermodynamics of the allosteric mechanism.References1. Kar, G., Keskin, O., Gursoy, A. & Nussinov, R. Curr Opin Pharmacol 10, 715–22 (2010).2. del Sol, A., Tsai, C.J., Ma, B. & Nussinov, R. Structure 17, 1042–1050 (2009).3. Pandini, A., Fornili, A., Kleinjung, J. BMC Bioinformatics 11, 97 (2010).

BackgroundBiomolecular motions play a key role in several biological functions: enzymatic activity, protein-protein interactions, ligand binding and allosteric regulation. Computational approaches, such as Molecular Dynamics (MD), are now routinely used to reproduce the intrinsic dynamics of proteins, but effective tools are still required to gain functional insight from the simulated data.Methods and ResultsWe previously suggested a method aimed at recovering the role of local conformational changes in functional motions. To this purpose we developed a Structural Alphabet (SA): a set of 25 canonical states of 4-residue protein fragments (Cα atoms only) describing the most probable local conformations in high-resolution protein structures [1]. The SA provides a mean for the coarse-grained annotation and processing of local conformations in a string format, which lends itself to a range of efficient sequence analysis algorithms. The SA has been successfully used in analysing local changes and allosteric signal transmission [2].Here we present GSATools [3], a set of SA-related tools interfacing with GROMACS for the analysis of conformational ensembles. GSATools is designed for the investigation of the conformational dynamics of local structures, the functional correlations between local and global motions, and the mechanisms of allosteric communication.ConclusionsGSATools is a free, easy-to-use and fully documented software for the analysis of conformational ensembles of proteins. The GSATools complement the GROMACS toolkit with a powerful set of analyses to detect, annotate and interpret local motions of functional relevance.1. Pandini A., Fornili A. & Kleinjung J. BMC Bioinformatics 11, 97 (2010).2. Pandini A., Fornili A., Fraternali F. & Kleinjung J., FASEB J., 26, 868 (2012).3. Pandini A., Fornili A., Fraternali F. & Kleinjung J., Bioinformatics, 29, 2053 (2013).