Structural Insights into the Human Mitochondrial Pyruvate Carrier Complexes.

Pyruvate metabolism requires the mitochondrial pyruvate carrier (MPC) proteins to transport pyruvate from the intermembrane space through the inner mitochondrial membrane to the mitochondrial matrix. The lack of the atomic structures of MPC hampers the understanding of the functional states of MPC and molecular interactions with the substrate or inhibitor. Here, we develop the de novo models of human MPC complexes and characterize the conformational dynamics of the MPC heterodimer formed by MPC1 and MPC2 (MPC1/2) by computational simulations. Our results reveal that functional MPC1/2 prefers to adopt an inward-open conformation, with the carrier open to the matrix side, whereas the outward-open states are less populated. The energy barrier for pyruvate transport in MPC1/2 is low enough, and the inhibitor UK5099 blocks the pyruvate transport by stably binding to MPC1/2. Notably, consistent with experimental results, the MPC1 L79H mutation significantly alters the conformations of MPC1/2 and thus fails for substrate transport. However, the MPC1 R97W mutation seems to retain the transport activity. The present de novo models of MPC complexes provide structural insights into the conformational states of MPC complexes and mechanistic understanding of interactions between the substrate/inhibitor and MPC proteins.

Identification of a New Allosteric Binding Site for Cocaine in Dopamine Transporter.

Dopamine (DA) transporter (DAT) is a major target for psychostimulant drugs of abuse such as cocaine that competitively binds to DAT, inhibits DA reuptake, and consequently increases synaptic DA levels. In addition to the central binding site inside DAT, the available experimental evidence suggests the existence of alternative binding sites on DAT, but detection and characterization of these sites are challenging by experiments alone. Here, we integrate multiple computational approaches to probe the potential binding sites on the wild-type Drosophila melanogaster DAT and identify a new allosteric site that displays high affinity for cocaine. This site is located on the surface of DAT, and binding of cocaine is primarily dominated by interactions with hydrophobic residues surrounding the site. We show that cocaine binding to this new site allosterically reduces the binding of DA/cocaine to the central binding pocket, and simultaneous binding of two cocaine molecules to a single DAT seems infeasible. Furthermore, we find that binding of cocaine to this site stabilizes the conformation of DAT but alters the conformational population and thereby reduces the accessibility by DA, providing molecular insights into the inhibitory mechanism of cocaine. In addition, our results indicate that the conformations induced by cocaine binding to this site may be relevant to the oligomerization of DAT, highlighting a potential role of this new site in modulating the function of DAT.

Extracellular gating of glucose transport through GLUT 1.

The ubiquitous glucose transporter 1 (GLUT1) is physiologically and pathologically relevant in energy metabolism of the CNS, skeletal muscles, cancer cells etc. Extensive experiments on GLUT1 produced thorough understandings of its expressions, functions, and structures which were recently resolved to atomic accuracy. However, theoretical understandings are still controversial about how GLUT1 facilitates glucose diffusion across the cell membrane. Molecular dynamics (MD) simulations of the current literature have GLUT1 embedded in a symmetric bilayer of a single lipid type. They provide atomistic illustrations of the alternating access theory (AAT), but the simulation results are inconsistent with the undisputed experimental data of kinetics showing rapid transport of glucose at near-physiological temperatures, high Arrhenius activation barrier in zero-trans uptake, and large trans-acceleration at sub-physiological temperatures. In this research, we embedded GLUT1 in an asymmetric bilayer of multiple lipids to better mimic the erythrocyte membrane. We ran unbiased MD simulations at 37 °C and at 5 °C and found a new mechanism of glucose transport via GLUT1: The extracellular (EC) gate opened wide for EC glucopyranose at 37 °C and, only in the presence of intracellular (IC) glucose, at 5 °C. In the absence of IC glucose at 5 °C, the EC gate opened narrowly for acyclic glucose, gating out glucopyranose. This EC-gating mechanism is simpler than AAT and yet it well explains for the rapid glucose transport at near-physiological temperatures and large trans-acceleration at sub-physiological temperatures. It also explains why zero-trans uptake (involving the pyranose-to-aldehyde transformation) has an Arrhenius barrier ∼20 kcal/mol higher than the equilibrium exchange transport.

Computing osmotic permeabilities of aquaporins AQP4, AQP5, and GlpF from near-equilibrium simulations.

Measuring or computing the single-channel permeability of aquaporins/aquaglyceroporins (AQPs) has long been a challenge. The measured values scatter over an order of magnitude but the corresponding Arrhenius activation energies converge in the current literature. Osmotic flux through an AQP was simulated as water current forced through the channel by kilobar hydraulic pressure or theoretically approximated as single-file diffusion. In this paper, we report large scale simulations of osmotic current under sub M gradient through three AQPs (water channels AQP4 and AQP5 and glycerol-water channel GlpF) using the mature particle mesh Ewald technique (PME) for which the established force fields have been optimized with known accuracy. These simulations were implemented with hybrid periodic boundary conditions devised to avoid the artifactitious mixing across the membrane in a regular PME simulation. The computed single-channel permeabilities at 5°C and 25°C are in agreement with recently refined experiments on GlpF. The Arrhenius activation energies extracted from our simulations for all the three AQPs agree with the in vitro measurements. The single-file diffusion approximations from our large-scale simulations are consistent with the current literature on smaller systems. From these unambiguous agreements among the in vitro and in silico studies, we observe the quantitative accuracy of the all-atom force fields of the current literature for water-channel biology. We also observe that AQP4, that is particularly rich in the central nervous system, is more efficient in water conduction and more temperature-sensitive than other water-only channels (excluding glycerol channels that also conduct water when not inhibited by glycerol).

Affinity and path of binding xylopyranose unto E. coli xylose permease.

Glucose transporters (GLUTs), expressed in all types of human cells, are responsible for the uptake of sugars as the primary energy source for the normal functions of good cells and for the abnormal growth of cancer cells. The E. coli xylose permease (XylE), a homologue of human GLUTs, has been investigated more thoroughly than other major facilitator proteins in the current literature. In this paper, we present a molecular dynamics (MD) study of an all-atom model system to elucidate the atomistic details and the free-energy landscape along the path of binding a xylopyranose (XYP) from the extracellular space to the inside of the transporter protein XylE. From the MD simulations, the Gibbs free energy of binding was found to be -4.4kcal/mol in agreement with the experimental value of -4.7kcal/mol. The accuracy of our study is further shown in the computed hydration energy of XYP of -14.6kcal/mol in comparison with the experimental data of -15.0kcal/mol. Along the binding path, the Gibbs free energy of the XYP-XylE complex first rises from zero in the dissociated state to approximately 4 kcal/mol in the transition state (when XylE slightly increases its opening toward the extracellular side to accommodate XYP) before dropping down to -9.0 kcal/mol in the bound state. These quantitative insights indicate the fast equilibration between the bound and the unbound states of XylE and XYP. They also serve as an atomistic-dynamic corroboration of the experimental conclusion that XylE is a high-affinity sugar transporter.

Elongation affinity, activation barrier, and stability of Aß42 oligomers/fibrils in physiological saline.

Amyloid-beta (Aß) peptides, Aß40 and the more neurotoxic Aß42, have been the subject of many research efforts for Alzheimer's disease. In two recent independent investigations, the atomistic structure of Aß42 fibril has been clearly established in the S-shaped conformation consisting of three ß-sheets stabilized by salt bridges formed between the Lys28 sidechain and the C-terminus of Ala42. This structure distinctively differs from the long-known structure of Aß40 in the ß-hairpin shaped conformation consisting of two ß-sheets. Recent in silico investigations based on all-atom models have reached closer agreement with the in vitro measurements of Aß40 thermodynamics. In this study, we present an in silico investigation of Aß42 thermodynamics. Using the established force field parameters in seven sets of all-atom simulations, we examined the stability of small Aß42 oligomers in physiological saline. We computed the elongation affinity of the S-shaped Aß42 fibril, reaching agreement with the experimental data. We also estimated the Arrhenius activation barrier along the elongation pathway (from the disordered conformation of a free Aß42 peptide to its S-shaped conformation on a fibril) that amounts to about 16 kcal/mol, which is consistent with the experimental data. Based on these quantitative agreements, we conclude that aggregation of Aß42 peptides into fibrils is thermodynamically slow without precipitation by extrinsic factors such as heparan sulfate proteoglycan and highlight the possibility to prevent Aß42 aggregation by eliminating some precipitation factors or by increasing competitive agents to capture and transport free Aß42 peptides from the cerebrospinal fluid.

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics.

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanics of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity---pulling out the ligand---closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification suffered by many sophisticated approaches in the literature.

Computing membrane-AQP5-phosphatidylserine binding affinities with hybrid steered molecular dynamics approach.

In order to elucidate how phosphatidylserine (PS6) interacts with AQP5 in a cell membrane, we developed a hybrid steered molecular dynamics (hSMD) method that involved: (1) Simultaneously steering two centers of mass of two selected segments of the ligand, and (2) equilibrating the ligand-protein complex with and without biasing the system. Validating hSMD, we first studied vascular endothelial growth factor receptor 1 (VEGFR1) in complex with N-(4-Chlorophenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (8ST), for which the binding energy is known from in vitro experiments. In this study, our computed binding energy well agreed with the experimental value. Knowing the accuracy of this hSMD method, we applied it to the AQP5-lipid-bilayer system to answer an outstanding question relevant to AQP5's physiological function: Will the PS6, a lipid having a single long hydrocarbon tail that was found in the central pore of the AQP5 tetramer crystal, actually bind to and inhibit AQP5's central pore under near-physiological conditions, namely, when AQP5 tetramer is embedded in a lipid bilayer? We found, in silico, using the CHARMM 36 force field, that binding PS6 to AQP5 was a factor of 3 million weaker than "binding" it in the lipid bilayer. This suggests that AQP5's central pore will not be inhibited by PS6 or a similar lipid in a physiological environment.

Ligand-modulated interactions between charged monolayer-protected Au144(SR)60 gold nanoparticles in physiological saline.

In order to determine how functionalized gold nanoparticles (AuNPs) interact in a near-physiological environment, we performed all-atom molecular dynamics simulations on the icosahedral Au144 nanoparticles each coated with a homogeneous set of 60 thiolates selected from one of these five (5) types: 11-mercapto-1-undecanesulfonate -SC11H22(SO3(-)), 5-mercapto-1-pentanesulfonate -SC5H10(SO3(-)), 5-mercapto-1-pentaneamine -SC5H10(NH3(+)), 4-mercapto-benzoate -SPh(COO(-)), or 4-mercapto-benzamide -SPh(CONH3(+)). These thiolates were selected to elucidate how the aggregation behavior of AuNPs depends on ligand parameters, including the charge of the terminal group (anionic vs. cationic), and its length and conformational flexibility. For this purpose, each functionalized AuNP was paired with a copy of itself, placed in an aqueous cell, neutralized by 120 Na(+)/Cl(-) counter-ions and salinated with a 150 mM concentration of NaCl, to form five (5) systems of like-charged AuNPs pairs in a saline. We computed the potential of mean force (the reversible work of separation) as a function of the intra-pair distance and, based on which, the aggregation affinities. We found that the AuNPs coated with negatively charged, short ligands have very high affinities. Structurally, a significant number of Na(+) counter-ions reside on a plane between the AuNPs, mediating the interaction. Each such ion forms a "salt bridge" (or "ionic bonds") to both of the AuNPs when they are separated by its diameter plus 0.2-0.3 nm. The positively charged AuNPs have much weaker affinities, as Cl(-) counter-ions form fewer and weaker salt bridges between the AuNPs. In the case of Au144(SC11H22(SO3(-)))60 pair, the flexible ligands fluctuate much more than the other four cases. The large fluctuations disfavor the forming of salt bridges between two AuNPs, but enable hydrophobic contact between the exposed hydrocarbon chains of the two AuNPs, which are subject to an effective attraction at a separation much greater than the AuNP diameter and involve a higher concentration of counter ions in the inter-pair space.

Glycerol modulates water permeation through Escherichia coli aquaglyceroporin GlpF.

Among aquaglyceroporins that transport both water and glycerol across the cell membrane, Escherichia coli glycerol uptake facilitator (GlpF) is the most thoroughly studied. However, one question remains: Does glycerol modulate water permeation? This study answers this fundamental question by determining the three-dimensional potential of mean force of glycerol along the permeation path through GlpF's conducting pore. There is a deep well near the Asn-Pro-Ala (NPA) motifs (6.5kcal/mol below the bulk level) and a barrier near the selectivity filter (10.1kcal/mol above the well bottom). This profile owes its existence to GlpF's perfect steric arrangement: The glycerol-protein van der Waals interactions are attractive near the NPA but repulsive elsewhere in the conducting pore. In light of the single-file nature of waters and glycerols lining up in GlpF's amphipathic pore, it leads to the following conclusion: Glycerol modulates water permeation in the µM range. At mM concentrations, GlpF is glycerol-saturated and a glycerol residing in the well occludes the conducting pore. Therefore, water permeation is fully correlated to glycerol dissociation that has an Arrhenius activation barrier of 6.5kcal/mol. Validation of this theory is based on the existent in vitro data, some of which have not been given the proper attention they deserved: The Arrhenius activation barriers were found to be 7kcal/mol for water permeation and 9.6kcal/mol for glycerol permeation; The presence of up to 100mM glycerol did not affect the kinetics of water transport with very low permeability, in apparent contradiction with the existent theories that predicted high permeability (0M glycerol).

Interaction between functionalized gold nanoparticles in physiological saline.

The interactions between functionalized noble-metal particles in an aqueous solution are central to applications relying on controlled equilibrium association. Herein, we obtain the potentials of mean force (PMF) for pair-interactions between functionalized gold nanoparticles (AuNPs) in physiological saline. These results are based upon >1000 ns experiments in silico of all-atom model systems under equilibrium and non-equilibrium conditions. Four types of functionalization are built by coating each globular Au144 cluster with 60 thiolate groups: GS-AuNP (glutathionate), PhS-AuNP (thiophenol), CyS-AuNP (cysteinyl), and p-APhS-AuNP (para-amino-thiophenol), which are, respectively, negatively charged, hydrophobic (neutral-nonpolar), hydrophilic (neutral-polar), and positively charged at neutral pH. The results confirm the behavior expected of neutral (hydrophilic or hydrophobic) particles in a dilute aqueous environment, however the PMF curves demonstrate that the charged AuNPs interact with one another in a unique way-mediated by H2O molecules and an electrolyte (Na(+), Cl(-))-in a physiological environment. In the case of two GS-AuNPs, the excess, neutralizing Na(+) ions form a mobile (or 'dynamic') cloud of enhanced concentration between the like-charged GS-AuNPs, inducing a moderate attraction (∼25 kT) between them. Furthermore, to a lesser degree, for a pair of p-APhS-AuNPs, the excess, neutralizing Cl(-) ions (less mobile than Na(+)) also form a cloud of higher concentration between the two like-charged p-APhS-AuNPs, inducing weaker yet significant attractions (∼12 kT). On combining one GS- with one p-APhS-AuNP, the direct, attractive Coulombic force is completely screened out while the solvation effects give rise to moderate repulsion between the two unlike-charged AuNPs.

Glycerol inhibits water permeation through Plasmodium falciparum aquaglyceroporin.

Plasmodium falciparum aquaglyceroporin (PfAQP) is a multifunctional membrane protein in the plasma membrane of P. falciparum, the parasite that causes the most severe form of malaria. The current literature has established the science of PfAQP's structure, functions, and hydrogen-bonding interactions but left unanswered the following fundamental question: does glycerol modulate water permeation through aquaglyceroporin that conducts both glycerol and water? This paper provides an affirmative answer to this question of essential importance to the protein's functions. On the basis of the chemical-potential profile of glycerol from the extracellular bulk region, throughout PfAQP's conducting channel, to the cytoplasmic bulk region, this study shows the existence of a bound state of glycerol inside aquaglyceroporin's permeation pore, from which the dissociation constant is approximately 14µM. A glycerol molecule occupying the bound state occludes the conducting pore through which permeating molecules line up in single file by hydrogen-bonding with one another and with the luminal residues of aquaglyceroporin. In this way, glycerol inhibits permeation of water and other permeants through aquaglyceroporin. The biological implications of this theory are discussed and shown to agree with the existent in vitro data. It turns out that the structure of aquaglyceroporin is perfect for the van der Waals interactions between the protein and glycerol to cause the existence of the bound state deep inside the conducting pore and, thus to play an unexpected but significant role in aquaglyceroporin's functions.

Quantitative characterization of the path of glucose diffusion facilitated by human glucose transporter 1.

Glucose transporter GLUT1 is ubiquitously expressed in the human body from the red cells to the blood-brain barrier to the skeletal muscles. It is physiologically relevant to understand how GLUT1 facilitates diffusion of glucose across the cell membrane. It is also pathologically relevant because GLUT1 deficiency causes neurological disorders and anemia and because GLUT1 overexpression fuels the abnormal growth of cancer cells. This article presents a quantitative investigation of GLUT1 based on all-atom molecular-dynamics (MD) simulations of the transporter embedded in lipid bilayers of asymmetric inner-and-outer-leaflet lipid compositions, subject to asymmetric intra-and-extra-cellular environments. This is in contrast with the current literature of MD studies that have not considered both of the aforementioned asymmetries of the cell membrane. The equilibrium (unbiased) dynamics of GLUT1 shows that it can facilitate glucose diffusion across the cell membrane without undergoing large-scale conformational motions. The Gibbs free-energy profile, which is still lacking in the current literature of GLUT1, quantitatively characterizes the diffusion path of glucose from the periplasm, through an extracellular gate of GLUT1, on to the binding site, and off to the cytoplasm. This transport mechanism is validated by the experimental data that GLUT1 has low water-permeability, uptake-efflux symmetry, and 10 kcal/mol Arrhenius activation barrier around 37 °C.

Effects of the N-terminal dynamics on the conformational states of human dopamine transporter.

Dopamine transporter mediates the neurotransmitter dopamine homeostasis in a sodium-dependent manner. The transport process involves an alternating access of a substrate to the extracellular and intracellular spaces, which is associated with different conformational states of the transporter. However, the underlying mechanism of modulation of the state transition remains elusive. Here we present a computational simulation study of human dopamine transporter to explore its two end states (outward-facing open and inward-facing open) that have not been determined experimentally. We show that the full-length transporter may tend to adopt the inward-facing open state in its free state. The binding of an amphetamine may not trap the transporter in the outward-facing open state with increasing length of the N-terminal. Furthermore, we identify distinct patterns in the interaction networks between the N-terminal and the intracellular region that could stabilize the state of the transporter, independent of substrate binding and phosphorylation. Our results reveal the essential role of the N-terminal dynamics in modulating the functional states of the dopamine transporter, providing molecular insights into the coupling of conformational transition and substrate passage in neurotransmitter transporters.

Tunnel connects lipid bilayer to occluded odorant-binding site of insect olfactory receptor.

MhOR5, an insect olfactory receptor (OR), has an occluded binding site for the odorant eugenol in both the open and closed states of the ion channel. We used atomistic molecular dynamics simulation (MD) and steered molecular dynamics to examine possible tunnels to the odorant binding site from the protein surface. Four high probability tunnels were identified in the MD results. Surprisingly, three of the tunnels connect the ligand binding site to the lipid bilayer. We found sharp 30%-50% increases or decreases in tunnel bottleneck areas over 70 nsec MD trajectories, both in the ligand-bound and unliganded OR structures. Steered MD showed that eugenol follows the tunnels to the protein surface, and the potential of mean force is quantitatively consistent with the known affinity of eugenol for MhOR5. We examined AlphaFold-generated models of 21 other insect ORs, and we found that 19 had odorant binding sites and tunnels in similar positions to MhOR5. The possibility of a tunnel between the odorant binding site and the lipid bilayer in insect ORs suggests new experiments to test molecular mechanisms for insect odorant reception.

Aquaglyceroporin AQP7's affinity for its substrate glycerol: Have we reached convergence in the computed values of glycerol-aquaglyceroporin affinity?

AQP7 is one of the four human aquaglyceroporins that facilitate glycerol transport across the cell membrane, a biophysical process that is essential in human physiology. Therefore, it is interesting to compute AQP7's affinity for its substrate (glycerol) with reasonable certainty to compare with the experimental data suggesting high affinity in contrast with most computational studies predicting low affinity. In this study aimed at computing the AQP7-glycerol affinity with high confidence, we implemented a direct computation of the affinity from unbiased equilibrium molecular dynamics (MD) simulations of three all-atom systems constituted with 0.16M, 4.32M, and 10.23M atoms, respectively. These three sets of simulations manifested a fundamental physics law that the intrinsic fluctuations of pressure in a system are inversely proportional to the system size (the number of atoms in it). These simulations showed that the computed values of glycerol-AQP7 affinity are dependent upon the system size (the inverse affinity estimations were, respectively, 47.3 mM, 1.6 mM, and 0.92 mM for the three model systems). In this, we obtained a lower bound for the AQP7-glycerol affinity (an upper bound for the dissociation constant). Namely, the AQP7-glycerol affinity is stronger than 1087/M (the dissociation constant is less than 0.92 mM). Additionally, we conducted hyper steered MD (hSMD) simulations to map out the Gibbs free-energy profile. From the free-energy profile, we produced an independent computation of the AQP7-glycerol dissociation constant being approximately 0.18 mM.

Association of sigma-1 receptor with dopamine transporter attenuates the binding of methamphetamine via distinct helix-helix interactions.

Dopamine transporter (DAT) and sigma-1 receptor (σ1R) are potential therapeutic targets to reduce the psychostimulant effects induced by methamphetamine (METH). Interaction of σ1R with DAT could modulate the binding of METH, but the molecular basis of the association of the two transmembrane proteins and how their interactions mediate the binding of METH to DAT or σ1R remain unclear. Here, we characterize the protein-ligand and protein-protein interactions at a molecular level by various theoretical approaches. The present results show that METH adopts a different binding pose in the binding pocket of σ1R and is more likely to act as an agonist. The relatively lower binding affinity of METH to σ1R supports the role of antagonists as inhibitors that protect against METH-induced effects. We demonstrate that σ1R could bind to Drosophila melanogaster DAT (dDAT) through interactions with either the transmembrane helix α12 or α5 of dDAT. Our results showed that the truncated σ1R displays stronger association with dDAT than the full-length σ1R. Although different helix-helix interactions between σ1R and dDAT lead to distinct effects on the dynamics of individual protein, both associations attenuate the binding affinity of METH to dDAT, particularly in the interactions with the helix α5 of dDAT. Together, the present study provides the first computational investigation on the molecular mechanism of coupling METH binding and the association of σ1R with dDAT.

Thermodynamic Integration in 3n Dimensions without Biases or Alchemy for Protein Interactions.

Thermodynamic integration (TI), a powerful formalism for computing Gibbs free energy, has been implemented for many biophysical processes with alchemical schemes that require delicate human efforts to choose/design biasing potentials for sampling the desired biophysical events and to remove their artifactitious consequences afterwards. Theoretically, an alchemical scheme is exact but practically, an unsophisticated implementation of this exact formula can cause error amplifications. Small relative errors in the input parameters can be amplified many times in their propagation into the computed free energy [due to subtraction of similar numbers such as (105 ± 5)‒(100 ± 5) = 5 ± 7]. In this paper, we present an unsophisticated implementation of TI in 3n dimensions (3nD) (n=1,2,3…) for the potential of mean force along a 3nD path connecting one state in the bound state ensemble to one state in the unbound state ensemble. Fluctuations in these 3nD are integrated in the bound and unbound state ensembles but not along the 3nD path. Using TI3nD, we computed the standard binding free energies of three protein complexes: trometamol in Salmonella effector SpvD (n=1), biotin-avidin (n=2), and Colicin E9 endonuclease with cognate immunity protein Im9 (n=3). We employed three different protocols in three independent computations of E9-Im9 to show TI3nD's robustness. We also computed the hydration energies of ten biologically relevant compounds (n=1 for water, acetamide, urea, glycerol, trometamol, ammonium and n=2 for erythritol, 1,3-propanediol, xylitol, biotin). Each of the 15 computations is accomplishable within one (for hydration) to ten (for E9-Im9) days on an inexpensive GPU workstation. The computed results all agree with the available experimental data.

Application of the Brown dynamics fluctuation-dissipation theorem to the study of Plasmodium berghei transporter protein PbAQP.

In this article, the Brownian dynamics fluctuation-dissipation theorem (BD-FDT) is applied to the study of transport of neutral solutes across the cellular membrane of Plasmodium berghei (Pb), a disease-causing parasite. Pb infects rodents and causes symptoms in laboratory mice that are comparable to human malaria caused by Plasmodium falciparum (Pf). Due to the relative ease of its genetic engineering, P. berghei has been exploited as a model organism for the study of human malaria. P. berghei expresses one type of aquaporin (AQP), PbAQP, and, in parallel, P. falciparum expresses PfAQP. Either PbAQP or PfAQP is a multifunctional channel protein in the plasma membrane of the rodent/human malarial parasite for homeostasis of water, uptake of glycerol, and excretion of some metabolic wastes across the cell membrane. This FDT-study of the channel protein PbAQP is to elucidate how and how strongly it interacts with water, glycerol, and erythritol. It is found that erythritol, which binds deep inside the conducting pore of PbAQP/PfAQP, inhibits the channel protein's functions of conducting water, glycerol etc. This points to the possibility that erythritol, a sugar substitute, may inhibit the malarial parasites in rodents and in humans.

Molecular determinant of substrate binding and specificity of cytochrome P450 2J2.

Cytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.