Atomistic MD Simulations Research Articles

Methionine‐aromatic Interaction as a Tool to Enhance Protein Stability

Nonbonded aromatic interactions play a significant role in stabilizing a folded protein structure(1, 2). Apart from well‐studied aromatic‐aromatic interactions in proteins, preferential proximity of sulfur containing cysteine and methionine sidechains and aromatic rings is also observed in protein hydrophobic cores and at the interfaces of protein‐protein complexes. Methionine‐aromatic motifs are examples of such S‐aromatic motifs that provide additional stabilization at a longer distance range over pure hydrophobic interactions(3). Although the idea of preferential association of aromatic amino acids has been successfully implemented for de novo design of peptides and proteins, the additional stability provided by met‐aromatic motif over pure hydrophobic interaction and its potential significance in designing a stabler protein fold has not been examined before. The present work shows the relevance of S‐aromatic interaction in increasing thermodynamic stability of a protein fold using Small Ubiquitin‐Like Modifier (SUMO) protein. We found that a highly conserved methionine‐phenylalanine motif in the core is a unique signature of SUMO but absent in other homologous Ubiquitin‐like (Ubl) protein folds. Using biophysical measurements and solution NMR based experiments, we found that the specific ‘up’ conformation between the methionine‐phenylalanine pair is critical to SUMO’s β‐grasp fold stability and its function(4). Given the additional stability provided by met‐aromatic motif over classical hydrophobic interaction, we engineered new S‐aromatic motifs in SUMO by replacing phenylalanine with other naturally occurring aromatic amino acids. Combining all atom MD simulations and solution NMR studies, we identify the energetically preferred interaction geometry of different met‐aromatic motifs inside the core. We found that the engineered SUMOs with increasing S‐aromatic interaction strength enhances the overall protein stability. Overall, our results reveal the potential role of S‐aromatic interactions as a design tool in enhancing thermodynamic stability of a protein fold.

Comparative structural and dynamics study of free and gRNA-bound FnCas9 and SpCas9 proteins

The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation, hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulation of free and gRNA bound forms of both the Cas9 proteins was performed, and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind the enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics, and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches.

Atomistic Model of Solute Transport across the Blood-Brain Barrier.

The blood–brain barrier remains a major roadblock to the delivery of drugs to the brain. While in vitro and in vivo measurements of permeability are widely used to predict brain penetration, very little is known about the mechanisms of passive transport. Detailed insight into interactions between solutes and cell membranes could provide new insight into drug design and screening. Here, we perform unbiased atomistic MD simulations to visualize translocation of a library of 24 solutes across a lipid bilayer representative of brain microvascular endothelial cells. A temperature bias is used to achieve steady state of all solutes, including those with low permeability. Based on free-energy surface profiles, we show that the solutes can be classified into three groups that describe distinct mechanisms of transport across the bilayer. Simulations down to 310 K for solutes with fast permeability were used to justify the extrapolation of values at 310 K from higher temperatures. Comparison of permeabilities at 310 K to experimental values obtained from in vitro transwell measurements and in situ brain perfusion revealed that permeabilities obtained from simulations vary from close to the experimental values to more than 3 orders of magnitude faster. The magnitude of the difference was dependent on the group defined by free-energy surface profiles. Overall, these results show that MD simulations can provide new insight into the mechanistic details of brain penetration and provide a new approach for drug discovery.

Potential efficacy of existing drug molecules against severe fever with thrombocytopenia syndrome virus: an in silico study

Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic disease caused by the SFTS virus (SFTSV). SFTS can be considered a life-threatening notifiable infectious disease. The unavailability of specific therapeutics encourages the investigation of potential efficacy of existing drugs against this infection. Drug repurposing was done by performing virtual screening of already established drug molecules followed by 100 ns molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area–based binding-energy calculation by targeting the SFTS L protein. On the basis of binding energy and protein–ligand interactions, top 10 promising hits were identified, showing stable binding with SFTS L protein. Further 100 ns atomistic MD simulation refined the hits from top 10 to top 4 with docking-based binding energy lesser than −8.0 kcal/mol toward the SFTS L protein and engaged in π–π interactions with pivotal amino acid residues. Various parameters and binding affinity of top 4 ligands towards L protein was computed. Ligand zaltoprofen exhibited best binding energy −220.095 kJ/mol. The present work is the first in silico study to assess bromfenac, cinchophen, elliptinium, and zaltoprofen; four promising hits against SFTS. Nonetheless, further proper biological evaluation is necessary to determine their efficacy against SFTS.

Allosteric Mechanism of Human Mitochondrial Phenylalanyl-tRNA Synthetase: An Atomistic MD Simulation and a Mutual Information-Based Network Study

Aminoacyl-tRNA synthetases (aaRSs), a family of ubiquitous and essential enzymes, can bind target tRNAs and catalyze the aminoacylation reaction in genetic code translation. In this work, we explore the dynamic properties and allosteric communication of human mitochondrial phenylalanyl-tRNA synthetase (hmPheRS) in free and bound states to understand the mechanisms of its tRNAPhe recognition and allostery using molecular dynamics simulations combined with the torsional mutual information-based network model. Our results reveal that hmPheRS's residue mobility and inter-residue motional coupling are significantly enhanced by tRNAPhe binding, and there occurs a strong allosteric communication which is critical for the aminoacylation reaction, suggesting the vital role of tRNAPhe binding in the enzyme's function. The identified signaling pathways mainly make the connections between the anticodon binding domain (ABD) and catalytic domain (CAD), as well as within the CAD composed of many functional fragments and active sites, revealing the co-regulation role of them to act coordinately and achieve hmPheRS's aminoacylation function. Besides, several key residues along the communication pathways are identified to be involved in mediating the coordinated coupling between anticodon recognition at the ABD and activation process at the CAD, showing their pivotal role in the allosteric network, which are well consistent with the experimental observation. This study sheds light on the allosteric communication mechanism in hmPheRS and can provide important information for the structure-based drug design targeting aaRSs.

Molecular simulations of lipid membrane partitioning and translocation by bacterial quorum sensing modulators.

Quorum sensing (QS) is a bacterial communication process mediated by both native and non-native small-molecule quorum sensing modulators (QSMs), many of which have been synthesized to disrupt QS pathways. While structure-activity relationships have been developed to relate QSM structure to the activation or inhibition of QS receptors, less is known about the transport mechanisms that enable QSMs to cross the lipid membrane and access intracellular receptors. In this study, we used atomistic MD simulations and an implicit solvent model, called COSMOmic, to analyze the partitioning and translocation of QSMs across lipid bilayers. We performed umbrella sampling at atomistic resolution to calculate partitioning and translocation free energies for a set of naturally occurring QSMs, then used COSMOmic to screen the water-membrane partition and translocation free energies for 50 native and non-native QSMs that target LasR, one of the LuxR family of quorum-sensing receptors. This screening procedure revealed the influence of systematic changes to head and tail group structures on membrane partitioning and translocation free energies at a significantly reduced computational cost compared to atomistic MD simulations. Comparisons with previously determined QSM activities suggest that QSMs that are least likely to partition into the bilayer are also less active. This work thus demonstrates the ability of the computational protocol to interrogate QSM-bilayer interactions which may help guide the design of new QSMs with engineered membrane interactions.

Energetics and Kinetics of Peptide-Lipid Membrane Interaction: A Molecular Dynamics Study

Quantitative characterization of the binding strength of peptides to lipid bilayers is crucial in understanding the molecular mechanism of cellular processes triggered by peptide-lipid interactions. In general, single molecule methods, such as high precision AFM based dynamic force spectroscopy, can be used to measure the dissociation force distribution, P(F), and the corresponding force dependent rupture rate, k(F), of individual peptides bound to a lipid bilayer. However, the interpretation of the results require extensive theoretical and computational modeling that implies the knowledge of the free energy profile (potential of mean force or PMF) of the system as a function of the separation between peptide and membrane surface. To this end, we have used all atom MD simulations, to calculate the PMF of three representative Wimley-White pentapeptides (Ac-WLXLL, with guest residues X=R,I and L) interacting with two different lipid bilayers (zwitterionic POPC and charged POPG). The PMF, as a function of the distance between the residue W and the plane of the membrane, was calculated by using (i) the equilibrium umbrella-sampling method, and (ii) the nonequilibrium FR method. Both methods yielded comparable PMFs. In addition, the FR method also permitted the calculation of the position dependent diffusion coefficient of the peptide. In each case, the values of the activation energy (free energy barrier) and activation length extracted from the PMFs appear to be comparable to previous experimental and computational studies. The obtained energetic and kinetic parameters have been successfully used to fit P(F) and k(F) measured in AFM experiments.

An atomistic view of solvent-free protein liquids: the case of Lipase A.

Solvent-free enzymes hold the promise of being able to deliver higher activity at elevated temperatures by virtue of them being not limited by the boiling point of the solvent. They have been realized in the liquid phase through a polymer surfactant coating on the protein surface. However, a clear understanding of intermolecular interactions, structure, dynamics, and the behaviour of the minuscule amount of water present in the solvent-free protein liquid is essential to enhance the activity of these biofluids. Using atomistic molecular dynamics simulations, we demonstrate that the scaled spatial correlations between proteins in the hybrid liquid phase of Lipase A enzymes are comparable to the inter-particle correlations in a noble gas fluid. The hydrophilic region of the surfactants forms a coronal layer around each enzyme which percolates throughout the liquid, while the hydrophobic parts are present as disjointed clusters. Inter-surfactant interactions, determined to be attractive and in the range of -200 to -300 kcal mol-1, stabilize the liquid state. While the protein retains its native state conformational dynamics in the solvent-free form, the fluxionality of its side chains is much reduced; at 333 K, the latter is found to be equivalent to that of the enzyme in an aqueous solution at 249 K. Despite the sluggishness of the solvent-free enzyme, some water molecules exhibit high mobility and transit between enzymes primarily via the interspersed hydrophilic regions. These microscopic insights offer ideas to improve substrate diffusion in the liquid to enable the enhancement of catalytic activity.

Dietary stigmastane-type saponins as promising dual-target directed inhibitors of SARS-CoV-2 proteases: a structure-based screening.

Despite the development of COVID-19 vaccines, at present, there is still no approved antiviral drug against the pandemic. The SARS-CoV-2 3-chymotrypsin-like proteases (S-3CLpro) and papain-like protease (S-PLpro) are essential for the viral proliferation cycle, hence attractive drug targets. Plant-based dietary components that have been extensively reported for antiviral activities may serve as cheap sources of preventive nutraceuticals and/or antiviral drugs. A custom-made library of 176 phytochemicals from five West African antiviral culinary herbs was screened for potential dual-target-directed inhibitors of S-3CLpro and S-PLpro in silico. The docking analysis revealed fifteen steroidal saponins (FSS) from Vernonia amygdalina with the highest binding tendency for the active sites of S-3CLpro and S-PLpro. In an optimized docking analysis, the FSS were further docked against four equilibrated conformers of the S-3CLpro and S-PLpro. Three stigmastane-type steroidal saponins (vernonioside A2, vernonioside A4 and vernonioside D2) were revealed as the lead compounds. These compounds interacted with the catalytic residues of both S-3CLpro and S-PLpro, thereby exhibiting dual inhibitory potential against these SARS-CoV-2 cysteine proteases. The binding free energy calculations further corroborated the static and optimized docking analysis. The complexed proteases with these promising phytochemicals were stable during a full atomistic MD simulation while the phytochemicals exhibited favourable physicochemical and ADMET properties, hence, recommended as promising inhibitors of SARS-CoV-2 cysteine proteases.

The Presence of a Wall Enhances the Probability for Ring‐Closing Metathesis: Insights from Classical Polymer Theory and Atomistic Simulations

AbstractThe probability distribution of chain ends meeting when one end of the polymer is fixed to a certain distance to a reflecting wall is investigated. For an ideal polymer chain the probability distribution can be evaluated analytically via classic polymer theory. These analytical predictions are compared to atomistic MD simulations of one tethered alkane chain close to the wall. The results demonstrate that a confining wall can lead to a significant increase in the return probability for the chain ends, and thus, can increase the occurrence of ring‐closing reactions. It is further demonstrated that the excess return probability shows a maximum at a certain distance, thereby yielding an optimal catalyst position in the ring‐closing reaction.

Homology modelling and virtual screening to explore potent inhibitors for MAP2K3 protein

MAP2K3 protein is mitogen-activated protein kinase belonging to the family of kinases involved in intracellular cell proliferation. The mammalian MAPK family that consists of ERK, p38 and JNK signalling pathway is showing a critical role in the regulation of cell proliferation. MAP2K3 and MAP2K6 are highly selective for p38 MAPKs which actively participate at check point controls and various stages of cell cycle at G0, G1/S and G2/M transitions by differential regulation of specific cyclin A or D1. In the present work, the 3D model of MAP2K3 protein is generated using comparative homology modelling techniques, minimized and validated. Active site of the protein is determined using various server tools and literature studies, for the prediction of important binding pockets to identify the putative active site. Virtual screening was carried out using chalcone library in Schrodinger suite to identify new lead molecules to knock down MAP2K3 target protein and inhibit cell proliferation. An Atomistic MD simulation of screened compound with MAP2K3 not only strengthens our findings but also identified the key interactions involved in protein-ligand complex in the dynamic environment.

Interdimeric Curvature in Tubulin-Tubulin Complexes Delineates the Microtubule-Destabilizing Properties of Plocabulin.

Plocabulin is a novel microtubule (MT) destabilizer agent with potent antineoplastic activity. This compound binds to the maytansine site at the longitudinal interface between tubulin dimers and exerts a hinge-like effect that disrupts normal microtubule assembly. Plocabulin has emerged as a valuable model for the rational design of novel MT destabilizers because of its unique structural and mechanistic features. To make progress on this matter, detailed molecular-level understanding of the ligand-protein interactions responsible for plocabulin association and the conformation and energetic effects arising from plocabulin binding on the longitudinal interaction between tubulin dimers must be provided. In this work, fully atomistic MD simulations and MM/GBSA binding free-energy calculations were used to examine the association of plocabulin to one or two tubulin dimers in longitudinal arrangement. Our results revealed that plocabulin binding is favored by the addition of a second tubulin dimer and that this ligand promotes the assembly of curved tetrameric arrangements with strengthened longitudinal interdimeric interactions compared to ligand-free systems. The applicability of these findings to the rational discovery of novel MT destabilizers was tested using MD and MM/GBSA calculations as filtering tools to narrow the results of virtual screening among an FDA-approved drug database. Our results confirmed that tight-binding ligands do not necessarily exert the expected conformational and energetic effects on longitudinal tubulin-tubulin interactions, which is a matter to consider in future design strategies.

Computational investigation of potential inhibitors of novel coronavirus 2019 through structure-based virtual screening, molecular dynamics and density functional theory studies

Despite the intensive research efforts towards antiviral drug against COVID-19, no potential drug or vaccines has not yet discovered. Initially, the binding site of COVID-19 main protease was predicted which located between regions 2 and 3. Structure-based virtual screening was performed through a hierarchal mode of elimination technique after generating a grid box. This led to the identification of five top hit molecules that were selected on the basis of docking score and visualization of non-bonding interactions. The docking results revealed that the hydrogen bonding and hydrophobic interactions are the major contributing factors in the stabilization of complexes. The docking scores were found between −7.524 and −6.711 kcal/mol indicating strong ligand-protein interactions. Amino acid residues Phe140, Leu141, Gly143, Asn142, Thr26, Glu166 and Thr190 (hydrogen bonding interactions) and Phe140, Cys145, Cys44, Met49, Leu167, Pro168, Met165, Val42, Leu27 and Ala191 (hydrophobic interactions) formed the binding pocket of COVID-19 main protease. From identified hits, ZINC13144609 and ZINC01581128 were selected for atomistic MD simulation and density functional theory calculations. MD simulation results confirm that the protein interacting with both hit molecules is stabilized in the chosen POPC lipid bilayer membrane. The presence of lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) in the hydrophobic region of the hit molecules leads to favorable ligand-protein contacts. The calculated pharmacokinetic descriptors were found to be in their acceptable range and therefore confirming their drug-like properties. Hence, the present investigation can serve as the basis for designing and developing COVID-19 inhibitors. Communicated by Ramaswamy H. Sarma

How global DNA unwinding causes non-uniform stress distribution and melting of DNA.

DNA unwinding is an important process that controls binding of proteins, gene expression and melting of double-stranded DNA. In a series of all-atom MD simulations on two DNA molecules containing a transcription start TATA-box sequence we demonstrate that application of a global restraint on the DNA twisting dramatically changes the coupling between helical parameters and the distribution of deformation energy along the sequence. Whereas only short range nearest-neighbor coupling is observed in the relaxed case, long-range coupling is induced in the globally restrained case. With increased overall unwinding the elastic deformation energy is strongly non-uniformly distributed resulting ultimately in a local melting transition of only the TATA box segment during the simulations. The deformation energy tends to be stored more in cytidine/guanine rich regions associated with a change in conformational substate distribution. Upon TATA box melting the deformation energy is largely absorbed by the melting bubble with the rest of the sequences relaxing back to near B-form. The simulations allow us to characterize the structural changes and the propagation of the elastic energy but also to calculate the associated free energy change upon DNA unwinding up to DNA melting. Finally, we design an Ising model for predicting the local melting transition based on empirical parameters. The direct comparison with the atomistic MD simulations indicates a remarkably good agreement for the predicted necessary torsional stress to induce a melting transition, for the position and length of the melted region and for the calculated associated free energy change between both approaches.

Analyzing Nucleosome Plasticity via Atomistic MD Simulations

Analyzing Nucleosome Plasticity via Atomistic MD Simulations

G-Protein-Coupled Receptors are Solvent-Swollen in the Functionally Active State

G-protein-coupled receptors (GPCRs) are transmembrane receptors that play critical roles in a number of physiological signaling processes. In the standard biochemical model, GPCRs behave as primarily agonist-dependent bimodal switches, with little influence of the surrounding medium. However, using the visual receptor rhodopsin as a model GPCR, we show that water drives rhodopsin to a partially disordered, solvent-swollen conformational ensemble upon light absorption, rendering the standard model obsolete. We placed rhodopsin under varying degrees of osmotic stress using polyethylene glycol solutes and investigated the activation equilibrium response using UV-visible spectroscopy. Our results show a flood of ∼80 water molecules into the rhodopsin interior during photoactivation, a result supported by atomistic MD simulations [1]. Furthermore, the osmolyte effects on rhodopsin activation are size-dependent: large osmolytes back shift the equilibrium to inactive metarhodopsin-I (MI), while small osmolytes forward-shift the equilibrium to active metarhodopsin-II (MII). We attribute these size effects to varying degrees of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation of rhodopsin. Besides osmotic pressure, the application of hydrostatic pressure also back shifts the metarhodopsin equilibrium but for fundamentally different reasons. Integrating the two force-based methods together with neutron scattering experiments [2] indicates that the active state of rhodopsin is more hydrated yet more locally collapsed. At the same time, the active GPCR undergoes volume fluctuations and solvent coupling, which give rise to greater thermal volume. The combined force-based results necessitate a new understanding of GPCR activation in which the surrounding soft matter is paramount in governing conformational energy landscapes. [1] N. Leioatts et al. (2014) Biochemistry 53, 376−385. [2] S.M.D.C. Perera et al. (2018) J. Phys. Chem. Lett.9, 7064−7071.

An Investigation of the Influenza Hemagglutinin Membrane Fusion Process using Microsecond-Level MD Simulations

Influenza virus hemagglutinin (HA) is a homotrimeric envelope protein that undergoes pH-triggered conformational changes in order to facilitate the fusion of the viral envelope with the endosomal membrane of the infected host cell. Previous experimental studies have outlined a mechanism for the conformational transitions that lead to membrane fusion. At low pH, the HA1 chains move away from the HA2 chains, which then undergo a series of conformational changes to expose the N-terminal fusion peptide. The HA2 chains then bend at a hinge region to form a hairpin shaped structure, thus bringing the viral envelope into contact with the endosomal membrane. While this model is well established, the conformational changes have never been observed in all-atom equilibrium MD simulations. Using a combination of equilibrium MD simulations and non-equilibrium SMD simulations, we have characterized the conformational dynamics of the membrane fusion process. Six models were simulated for 2.4 microseconds each. By modifying the protonation state of a conserved histidine in the HA2 hinge region, we have successfully simulated the pH-mediated conformational changes that occur when HA1 moves away from HA2. Our atomistic MD simulations confirmed that the HA1 chain associated with an HA2 chain moves away only when the histidine is protonated. Using non-equilibrium pulling simulations, we have also modeled the complex conformational and secondary-structural transitions that lead to the uncoiling of HA2 and the exposure of the fusion peptide. In addition to the conformational changes that occur within hemagglutinin, we have independently modeled the fusion of the viral envelope with the host cell endosomal membrane. For the very first time, we have studied the conformational dynamics of the entire HA-membrane complex using microsecond-level atomistic MD simulations and non-equilibrium simulations.

Effects of an atomistic modeling approach on predicted mechanical properties of glassy polymers via molecular dynamics

Glassy polymers are utilized in numerous applications ranging from light-weight structural materials to membranes for industrial gas separation. In this study, we quantify the ability of non-equilibrium molecular dynamics (NEMD) simulations to predict mechanical properties of glassy polymers based on different modeling approaches: force field selection, number of polymer chains in the simulation cell, and polymer builder algorithm. The polymers analyzed in this work are poly(methyl methacrylate) (PMMA), poly(propylene) (PP), and a polymer of intrinsic microporosity (PIM-1). PMMA samples were synthesized in silico using three different methods: continuous configurational biased Monte Carlo, pseudo-self-avoiding random walk, and a generalized simulated polymerization approach. Following the application of a consistent equilibration approach for each PMMA sample, stress–strain data from simulated tensile testing revealed that the choice of polymer builder algorithm or number of chains comprising the simulation cell did not have significant impact on the predicted elastic modulus. Force field selection effects have been analyzed by applying generalized force fields (GAFF and DREIDING) to each PMMA and PP sample and were found to be the most influencing factor studied. For these polymers, it was found that the DREIDING force field provides excellent agreement with experimental tensile moduli, ∼3.25 GPa and ∼1.53 GPa for PMMA and PP, respectively, while GAFF provides a systematic overestimation of the modulus by approximately 1.0 GPa. However, in the case of the PIM-1 model, with previously validated bonded interactions described by GAFF and non-bonded parameters from the TraPPE force field, the tensile modulus was predicted to be 1.23 GPa, which is well within the range of measured experimental values. Altogether, the simulations performed in this study illustrate the capabilities of atomistic MD simulations to predict the elastic modulus of glassy polymers and highlight energetic potential terms to consider for force field validation.

Mechanism of Molecular Oxygen Diffusion in a Hypoxia-Sensing Prolyl Hydroxylase Using Multiscale Simulation.

The chronic response of animals to hypoxia is mediated by the αβ-heterodimeric hypoxia-inducible transcription factors (α,β-HIFs) which upregulate the expression of sets of genes that work to ameliorate the effects of limiting dioxygen. The HIF prolyl hydroxylase domain enzymes (PHDs) are Fe(II)- and 2-oxoglutarate-dependent oxygenases that act as hypoxia-sensing components of the HIF system: prolyl-hydroxylation signals for dioxgen availability-dependent HIF-α degradation via the ubiquitin proteasome system. The unusual kinetic properties of the PHDs, in particular a high Km for dioxygen and slow reaction with dioxygen, are proposed to enable their hypoxia-sensing role. An understanding of how dioxygen is delivered to, and binds at, the active site of the PHDs is important for the development of a chemical understanding of the hypoxic response. We employed a combined multiscale approach involving classical atomistic equilibrium and nonequilibrium MD simulations combined with QM/MM trajectories to investigate dioxygen diffusion to, and binding at, the active site in the PHD2.Fe(II).2OG.HIF substrate complex; PHD2 is the most important of the three human PHDs. The transport of dioxygen to the active site is described; dioxygen transport follows a single well-defined hydrophobic tunnel, formed from both enzyme and substrate elements to reach the PHD2 active site. The results provide estimates for rate constants that define a diffusion-reaction model for dioxygen:PHD2 interactions; in combination with reported biophysical analyses they provide chemical insight into the basis of the slow reaction of PHD2 with dioxygen. They imply that the reversible binding of dioxygen is central to the hypoxia-sensing capacity of the PHDs and that different PHD HIF-α substrate combinations might have different dioxygen sensitivity profiles. The extent of HIF-α substrate prolyl hydroxylation, which signals for subsequent HIF-α degradation, may thus be a manifestation of the equilibrium between dioxygen in bulk solution and dioxygen bound to the PHD2.Fe.2OG.HIF-α substrate complex.

Energy flow and intersubunit signalling in GSAM: A non-equilibrium molecular dynamics study

Non-equilibrium molecular dynamics simulations of vibrational energy flow induced by the imposition of a thermal gradient have been performed on the μ2-dimeric enzyme glutamate-1-semialdehyde aminomutase (GSAM), the key enzyme in the biosynthesis of chlorophyll, in order to identify energy transport pathways and to elucidate their role as potential allosteric communication networks for coordinating functional dynamics, specifically the negative cooperativity observed in the motion of the two active site gating loops. Fully atomistic MD simulations of thermal diffusion were executed with a GROMACS simulation package on a fully solvated GSAM enzyme by heating various active site target ligands (initially, catalytic intermediates and cofactors) to 300K while holding the remainder of the protein and the solvent bath at 10K and monitoring the temperature T(t) of all the enzyme residues as a function of time over a 1ns observation window. Energy is observed to be deposited in a relatively small number of discrete chains of residues most of which contribute to specific structural or biochemical functionality. Thermal linkages between all thermally active chains were established by isolating a specific pair of chains and performing a thermal diffusion simulation on the pair, one held at 300K and the other at 10K, with the rest of the protein frozen in its initial atomic configuration and thus thermally unresponsive. Proceeding in this way, it was possible to map out multiple pathways of vibrational energy flow leading from one of the active sites through a network of contiguous residues, many of which were evolutionarily conserved and linked by hydrogen bonds, into the other active site and ultimately to the other gating loop.