Allosteric Communication Pathways Research Articles

Intermolecular Protein-Water Coupling Impedes the Coupling Between the Amide A and Amide I Mode in Interfacial Proteins.

The coupling between different vibrational modes in proteins is essential for chemical dynamics and biological functions and is linked to the propagation of conformational changes and pathways of allosteric communication. However, little is known about the influence of intermolecular protein-H2O coupling on the vibrational coupling between amide A (NH) and amide I (C═O) bands. Here, we investigate the NH/CO coupling strength in various peptides with different secondary structures at the lipid cell membrane/H2O interface using femtosecond time-resolved sum frequency generation vibrational spectroscopy (SFG-VS) in which a femtosecond infrared pump is used to excite the amide A band, and SFG-VS is used to probe transient spectral evolution in the amide A and amide I bands. Our results reveal that the NH/CO coupling strength strongly depends on the bandwidth of the amide I mode and the coupling of proteins with water molecules. A large extent of protein-water coupling significantly reduces the delocalization of the amide I mode along the peptide chain and impedes the NH/CO coupling strength. A large NH/CO coupling strength is found to show a strong correlation with the high energy transfer rate found in the light-harvesting proteins of green sulfur bacteria, which may understand the mechanism of energy transfer through a molecular system and assist in controlling vibrational energy transfer by engineering the molecular structures to achieve high energy transfer efficiency.

Allosteric Activation of RhoA Complexed with p115-RhoGEF Deciphered by Conformational Dynamics.

The Ras homologue family member A (RhoA) is a member of the Rho family, a subgroup of the Ras superfamily. RhoA interacts with the 115 kDa guanine nucleotide exchange factor (p115-RhoGEF), which assists in activation and binding with downstream effectors. Here, we use molecular dynamics (MD) simulations and essential dynamics analysis of the inactive RhoA-GDP and active RhoA-GTP, when bound to p115-RhoGEF to decipher the mechanism of RhoA activation at the structural level. We observe that inactive RhoA-GDP maintains its position near the catalytic site on the Dbl homology (DH) domain of p115-RhoGEF through the interaction of its Switch I region with the DH domain. We further show that the active RhoA-GTP is engaged in more interactions with the p115-RhoGEF membrane-bound Pleckstrin homology (PH) domain as compared to RhoA-GDP. We hypothesize that the role of the interactions between the active RhoA-GTP and the PH domain is to help release it from the DH domain upon activation. Our results support this premise, and our simulations uncover the beginning of this process and provide structural details. They also point to allosteric communication pathways that take part in RhoA activation to promote and strengthen the interaction between the active RhoA-GTP and the PH domain. Allosteric regulation also occurs among other members of the Rho superfamily. Collectively, we suggest that in the activation process, the role of the RhoA-GTP interaction with the PH domain is to release RhoA-GTP from the DH domain after activation, making it available to downstream effectors.

Dynamic Insights into the Self-Activation Pathway and Allosteric Regulation of the Orphan G-Protein-Coupled Receptor GPR52.

Within over 800 members of G-protein-coupled receptors, there are numerous orphan receptors whose endogenous ligands are largely unknown, providing many opportunities for novel drug discovery. However, the lack of an in-depth understanding of the intrinsic working mechanism for orphan receptors severely limits the related rational drug design. The G-protein-coupled receptor 52 (GPR52) is a unique orphan receptor that constitutively increases cellular 5'-cyclic adenosine monophosphate (cAMP) levels without binding any exogenous agonists and has been identified as a promising therapeutic target for central nervous system disorders. Although recent structural biology studies have provided snapshots of both active and inactive states of GPR52, the mechanism of the conformational transition between these states remains unclear. Here, an acceptable self-activation pathway for GPR52 was proposed through 6 μs Gaussian accelerated molecular dynamics (GaMD) simulations, in which the receptor spontaneously transitions from the active state to that matching the inactive crystal structure. According to the three intermediate states of the receptor obtained by constructing a reweighted potential of mean force, how the allosteric regulation occurs between the extracellular orthosteric binding pocket and the intracellular G-protein-binding site is revealed. Combined with the independent gradient model, several important microswitch residues and the allosteric communication pathway that directly links the two regions are both identified. Transfer entropy calculations not only reveal the complex allosteric signaling within GPR52 but also confirm the unique role of ECL2 in allosteric regulation, which is mutually validated with the results of GaMD simulations. Overall, this work elucidates the allosteric mechanism of GPR52 at the atomic level, providing the most detailed information to date on the self-activation of the orphan receptor.

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism.

Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug-drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

Allosteric Hotspots in the Main Protease of SARS-CoV-2

Inhibiting the main protease of SARS-CoV-2 is of great interest in tackling the COVID-19 pandemic caused by the virus. Most efforts have been centred on inhibiting the binding site of the enzyme. However, considering allosteric sites, distant from the active or orthosteric site, broadens the search space for drug candidates and confers the advantages of allosteric drug targeting. Here, we report the allosteric communication pathways in the main protease dimer by using two novel fully atomistic graph-theoretical methods: Bond-to-bond propensity, which has been previously successful in identifying allosteric sites in extensive benchmark data sets without a priori knowledge, and Markov transient analysis, which has previously aided in finding novel drug targets in catalytic protein families. Using statistical bootstrapping, we score the highest ranking sites against random sites at similar distances, and we identify four statistically significant putative allosteric sites as good candidates for alternative drug targeting.

The Role of Conformational Dynamics and Allostery in the Control of Distinct Efficacies of Agonists to the Glucocorticoid Receptor.

Glucocorticoid receptor (GR) regulates various cellular functions. Given its broad influence on metabolic activities, it has been the target of drug discovery for decades. However, how drugs induce conformational changes in GR has remained elusive. Herein, we used five GR agonists (dex, AZ938, pred, cor, and dibC) with different efficacies to investigate which aspect of the ligand induced the differences in efficacy. We performed molecular dynamics simulations on the five systems (dex-, AZ938-, pred-, cor-, and dibC-bound systems) and observed a distinct discrepancy in the conformation of the cofactor TIF2. Moreover, we discovered ligand-induced differences regarding the level of conformational changes posed by the binding of cofactor TIF2 and identified a pair of essential residues D590 and T39. We further found a positive correlation between the efficacies of ligands and the interaction of the two binding pockets’ domains, where D590 and T739 were involved, implying their significance in the participation of allosteric communication. Using community network analysis, two essential communities containing D590 and T739 were identified with their connectivity correlating to the efficacy of ligands. The potential communication pathways between these two residues were revealed. These results revealed the underlying mechanism of allosteric communication between the ligand-binding and cofactor-binding pockets and identified a pair of important residues in the allosteric communication pathway, which can serve as a guide for future drug discovery.

Prediction of allosteric communication pathways in proteins.

Allostery in proteins is an essential phenomenon in biological processes. In this article, we present a computational model to predict paths of maximum information transfer between active and allosteric sites. In this information theoretic study, we use mutual information as the measure of information transfer, where transition probability of information from one residue to its contacting neighbors is proportional to the magnitude of mutual information between the two residues. Starting from a given residue and using a Hidden Markov Model, we successively determine the neighboring residues that eventually lead to a path of optimum information transfer. The Gaussian approximation of mutual information between residue pairs is adopted. The limits of validity of this approximation are discussed in terms of a nonlinear theory of mutual information and its reduction to the Gaussian form. Predictions of the model are tested on six widely studied cases, CheY Bacterial Chemotaxis, B-cell Lymphoma extra-large (Bcl-xL), Human proline isomerase cyclophilin A (CypA), Dihydrofolate reductase (DHFR), HRas GTPase and Caspase-1. The communication transmission rendering the propagation of local fluctuations from the active sites throughout the structure in multiple paths correlate well with the known experimental data. Distinct paths originating from the active site may likely represent a multi functionality such as involving more than one allosteric site and/or pre-existence of some other functional states. Our model is computationally fast and simple and can give allosteric communication pathways, which are crucial for the understanding and control of protein functionality. Supplementary data are available at Bioinformatics online.

Subsets of Slow Dynamic Modes Reveal Global Information Sources as Allosteric Sites

Allostery is a key biological control mechanism, and dynamic information flow provides a perspective to describe allosteric interactions in causal relationships. Here, as a novel implementation of the Gaussian Network Model (GNM) based Transfer Entropy (TE) calculations, we show that the dissection of dynamic information into subsets of slow dynamic modes discloses different layers of multi-directional allosteric pathways inherent in a given protein structure. In these subsets of slow modes, the degree of collectivity (Col) in the information transfer of residues with their TE values (TECol score) identifies distinct residues as powerful effectors, global information sources; showing themselves with a high dynamic capacity to collectively disseminate information to others. As exemplified on aspartate transcarbamoylase (ATCase), Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), and human transient receptor potential melastatin 2 (TRPM2) along with a dataset of 20 proteins, these specific residues are associated with known active and allosteric sites. These information source residues, which collectively control others and lead allosteric communication pathways, hint at plausible binding sites for structure-based rational drug design.

Ligand‐specific mechanisms of allosteric regulation in FXR

Farnesoid X receptor (FXR) is a member of the nuclear receptor family of ligand‐regulated transcription factors that regulates bile acid, lipid and glucose metabolism. Bile acids are the endogenous FXR ligands. Bile acid binding to FXR induces association with FXR response element sequences (FXREs), and promotes selective recruitment of coregulator proteins via induced structural changes in the activation function AF‐2 surface, i.e. the site of coregulator binding. However, there is a poor understanding of how FXR is allosterically regulated by bile acid, which acts as a molecular switch to initiate critical long‐range of transcriptional events. The goal of this work is to understand how minor structural modifications in bile acids give rise to differential FXR transcriptional activity. We used dual luciferase assays to determine how bile acids modulate FXR DNA binding preferences for a a wide range of FXREs. We also used molecular dynamics (MD) simulations to study the effect of the bile acids on AF‐2 signaling. We have identified key residues that mediate specific allosteric communication pathways in FXR, and the mutations of the key residues in FXR‐LBD affects bile acid‐specific FXR transcriptional activities, confirming their roles in ligand‐AF‐2 allostery. These findings can further our understanding of how bile acids impact FXR promoter selectivity and coregulator recruitment.

From Distinct to Differential Conformational Dynamics to Map Allosteric Communication Pathways in Proteins.

Initiation of biological processes involving protein-ligand binding, transient protein-protein interactions, or amino acid modifications alters the conformational dynamics of proteins. Accompanying these biological processes are ensuing coupled atomic level conformational changes within the proteins. These conformational changes collectively connect multiple amino acid residues at distal allosteric, binding, and/or active sites. Local changes due to, for example, binding of a regulatory ligand at an allosteric site initiate the allosteric regulation. The allosteric signal propagates throughout the protein structure, causing changes at distal sites, activating, deactivating, or modifying the function of the protein. Hence, dynamical responses within protein structures to stimuli contain critical information on protein function. In this Perspective, we examine the description of allosteric regulation from protein dynamical responses and associated alternative and emerging computational approaches to map allosteric communication pathways between distal sites in proteins at the atomic level.

Structural evolution of the ancient enzyme, dissimilatory sulfite reductase.

Dissimilatory sulfite reductase is an ancient enzyme that has linked the global sulfur and carbon biogeochemical cycles since at least 3.47 Gya. While much has been learned about the phylogenetic distribution and diversity of DsrAB across environmental gradients, far less is known about the structural changes that occurred to maintain DsrAB function as the enzyme accompanied diversification of sulfate/sulfite reducing organisms (SRO) into new environments. Analyses of available crystal structures of DsrAB from Archaeoglobus fulgidus and Desulfovibrio vulgaris, representing early and late evolving lineages, respectively, show that certain features of DsrAB are structurally conserved, including active siro-heme binding motifs. Whether such structural features are conserved among DsrAB recovered from varied environments, including hot spring environments that host representatives of the earliest evolving SRO lineage (e.g., MV2-Eury), is not known. To begin to overcome these gaps in our understanding of the evolution of DsrAB, structural models from MV2.Eury were generated and evolutionary sequence co-variance analyses were conducted on a curated DsrAB database. Phylogenetically diverse DsrAB harbor many conserved functional residues including those that ligate active siro-heme(s). However, evolutionary co-variance analysis of monomeric DsrAB subunits revealed several False Positive Evolutionary Couplings (FPEC) that correspond to residues that have co-evolved despite being too spatially distant in the monomeric structure to allow for direct contact. One set of FPECs corresponds to residues that form a structural path between the two active siro-heme moieties across the interface between heterodimers, suggesting the potential for allostery or electron transfer within the enzyme complex. Other FPECs correspond to structural loops and gaps that may have been selected to stabilize enzyme function in different environments. These structural bioinformatics results suggest that DsrAB has maintained allosteric communication pathways between subunits as SRO diversified into new environments. The observations outlined here provide a framework for future biochemical and structural analyses of DsrAB to examine potential allosteric control of this enzyme.

Regulation of Cholesterol Binding to the Receptor Patched1 by its interactions With the Ligand Sonic Hedgehog (Shh).

The Hedgehog (Hh) signaling pathway is essential in cell development and regeneration, which is activated by the ligand Sonic hedgehog (Shh). The binding of Shh to its receptor Patched1 (PTCH1) releases the inhibitory effect on the downstream protein Smoothened (SMO), a G-protein-coupled-receptor (GPCR) protein. Cholesterol was supposed to function as a secondary messenger between PTCH1 and SMO. However, the molecular mechanism of this regulation process is still unclear. Therefore, microsecond coarse-grained molecular dynamics simulations were performed to investigate the protein-lipid interactions of the PTCH1 monomer and dimer-Shh complex. It was observed that the binding of cholesterols to the monomer is more stable than that to the dimer-Shh complex. It is regulated by the enrichment of Ganglioside lipids around proteins and the conformation of Y446, a residue in the sterol-sensing domain (SSD). The regulation of Shh on the dynamics of PTCH1 was further analyzed to explore the allosteric communication pathways between the Shh and the SSD. Our study provides structural and dynamic details of an additional perspective on the regulation of Hh signaling pathway through the lipid micro-environments of PTCH1.

Residue-Residue Contact Changes during Functional Processes Define Allosteric Communication Pathways.

Allosteric regulation plays a central role in orchestrating diverse cellular processes. A prerequisite for allostery is a flexible biomolecule within which two distal sites can communicate via concerted or sequential conformational changes. We introduce a computational method to elucidate allosteric communication pathways, comprising critical allosteric residues, in biomolecules by taking advantage of conformational changes during a functional process. Conformational changes are modeled explicitly since they modulate the network of residue-residue interactions, which could propagate allosteric signals between two or more distal sites. The method implements the suboptimal path analysis in the framework of the difference contact network analysis or dCNA. The method identifies key experimentally verified allosteric residues in imidazole glycerol phosphate synthase (IGPS), a well-studied allosteric protein system. By contrast, some of the most important allosteric residues are not captured using methods that do not consider conformational changes, such as those that solely rely on examining the individual bound or unbound state of the protein. Using the dCNA path analysis along with conventional analyses, we gain several new biological insights into IGPS. Interestingly, different binding processes in the thermodynamic cycle generally use a similar group of residues in defining the allosteric communication pathways, with some residues being more specific to a certain binding process. We also observed that the fine-tuning of allosteric coupling depends on the strength of effector binding. Our results are robust against small variations of parameters and details of the network construction. The dCNA path analysis method is general and can be easily applied to diverse allosteric systems.

Potential allosteric sites captured in glycolytic enzymes via residue-based network models: Phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase

Likelihood of new allosteric sites for glycolytic enzymes, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GADPH) and pyruvate kinase (PK) was evaluated for bacterial, parasitic and human species. Allosteric effect of a ligand binding at a site was revealed on the basis of low-frequency normal modes via Cα-harmonic residue network model. In bacterial PFK, perturbation of the proposed allosteric site outperformed the known allosteric one, producing a high amount of stabilization or reduced dynamics, on all catalytic regions. Another proposed allosteric spot at the dimer interface in parasitic PFK exhibited major stabilization effect on catalytic regions. In parasitic GADPH, the most desired allosteric response was observed upon perturbation of its tunnel region which incorporated key residues for functional regulation. Proposed allosteric site in bacterial PK produced a satisfactory allosteric response on all catalytic regions, whereas in human and parasitic PKs, a partial inhibition was observed. Residue network model based solely on contact topology identified the ‘hub residues’ with high betweenness tracing plausible allosteric communication pathways between distant functional sites. For both bacterial PFK and PK, proposed sites accommodated hub residues twice as much as the known allosteric site. Tunnel region in parasitic GADPH with the strongest allosteric effect among species, incorporated the highest number of hub residues. These results clearly suggest a one-to-one correspondence between the degree of allosteric effect and the number of hub residues in that perturbation site, which increases the likelihood of its allosteric nature.

Mechanistic Insights into the Allosteric Inhibition of Androgen Receptors by Binding Function 3 Antagonists from an Integrated Molecular Modeling Study.

An androgen receptor (AR) is an intensively studied treatment target for castration-resistant prostate cancer that is irresponsive to conventional antiandrogen therapeutics. Binding function 3 (BF3) inhibitors with alternative modes of action have emerged as a promising approach to overcoming antiandrogen resistance. However, how these BF3 inhibitors modulate AR function remains elusive, hindering the development of BF3-targeting agents. Here, we performed an integrated computational study to interrogate the binding mechanism of several known BF3 inhibitors with ARs. Our results show that the inhibitory effect of the BF3 antagonists arises from their allosteric modulation of the activation function (AF2) site, which alters the dynamic coupling between the BF3 and AF2 sites as well as the AF2-coactivator (SRC2-3) interaction. Moreover, the per-residue binding energy analyses reveal the "anchor" role of the linker connecting the phenyl ring and benzimidazole/indole in these BF3 inhibitors. Furthermore, the allosteric driver-interacting residues are found to include both "positive", e.g., Phe673 and Asn833, and "negative" ones, e.g., Phe826, and the differential interactions with these residues provide an explanation why stronger binding does not necessarily result in higher inhibitory activities. Finally, our allosteric communication pathway analyses delineate how the allosteric signals triggered by BF3 binding are propagated to the AF2 pocket through multiple short- and/or long-ranged transmission pathways. Collectively, our combined computational study provides a comprehensive structural mechanism underlying how the selected set of BF3 inhibitors modulate AR function, which will help guide future development of BF3 antagonists.

Through bonds or contacts? Mapping protein vibrational energy transfer using non-canonical amino acids

Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the β-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.

Allosteric modulation of cardiac myosin mechanics and kinetics by the conjugated omega‐7,9 trans‐fat rumenic acid

Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of β-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the β-cardiac myosin-2 motor domain with an EC50 of 200nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine β-cardiac myosin, the EC50 for rumenic acid varies from 10.5μM in the absence of actin to 200nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of β-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5μM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

Untangling Dual-Targeting Therapeutic Mechanism of Epidermal Growth Factor Receptor (EGFR) Based on Reversed Allosteric Communication.

Dual-targeting therapeutics by coadministration of allosteric and orthosteric drugs is drawing increased attention as a revolutionary strategy for overcoming the drug-resistance problems. It was further observed that the occupation of orthosteric sites by therapeutics agents has the potential to enhance allosteric ligand binding, which leads to improved potency of allosteric drugs. Epidermal growth factor receptor (EGFR), as one of the most critical anti-cancer targets belonging to the receptor tyrosine kinase family, represents a quintessential example. It was revealed that osimertinib, an ATP-competitive covalent EGFR inhibitor, remarkably enhanced the affinity of a recently developed allosteric inhibitor JBJ-04-125-02 for EGFRL858R/T790M. Here, we utilized extensive large-scale molecular dynamics simulations and the reversed allosteric communication to untangle the detailed molecular underpinning, in which occupation of osimertinib at the orthosteric site altered the overall conformational ensemble of EGFR mutant and reshaped the allosteric site via long-distance signaling. A unique intermediate state resembling the active conformation was identified, which was further stabilized by osimertinib loading. Based on the allosteric communication pathway, we predicted a novel allosteric site positioned around K867, E868, H893, and K960 within the intermediate state. Its correlation with the orthosteric site was validated by both structural and energetic analysis, and its low sequence conservation indicated the potential for selective targeting across the human kinome. Together, these findings not only provided a mechanistic basis for future clinical application of the dual-targeting therapeutics, but also explored an innovative perception of allosteric inhibition of tyrosine kinase signaling.

Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics.

Understanding allostery in enzymes and tools to identify it offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways in two prototypical class A β-lactamases, TEM-1 and KPC-2, which are important determinants of antibiotic resistance. The nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. Propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more of clinically relevant amino acid substitutions map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover, and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

P53 Is Potentially Regulated by Cyclophilin D in the Triple-Proline Loop of the DNA Binding Domain.

The multifunctional protein p53 is the central molecular sensor of cellular stresses. The canonical function of p53 is to transcriptionally activate target genes in response to, for example, DNA damage that may trigger apoptosis. Recently, p53 was also found to play a role in the regulation of necrosis, another type of cell death featured by the mitochondrial permeability transition (mPT). In this process, p53 directly interacts with the mPT regulator cyclophilin D, the detailed mechanism of which however remains poorly understood. Here, we report a comprehensive computational investigation of the p53-cyclophilin D interaction using molecular dynamics simulations and associated analyses. We have identified the specific cyclophilin D binding site on p53 that is located at proline 151 in the DNA binding domain. As a peptidyl-prolyl isomerase, cyclophilin D binds p53 and catalyzes the cis-trans isomerization of the peptide bond preceding proline 151. We have also characterized the effect of such an isomerization and found that the p53 domain in the cis state is overall more rigid than the trans state except for the local region around proline 151. Dynamical changes upon isomerization occur in both local and distal regions, indicating an allosteric effect elicited by the isomerization. We present potential allosteric communication pathways between proline 151 and distal sites, including the DNA binding surface. Our work provides, for the first time, a model for how cyclophilin D binds p53 and regulates its activity by switching the configuration of a specific site.