Dynamics of CYP51: implications for function and inhibitor design

Sterol 14α‐demethylase (cytochrome P450 family 51 (CYP51)) is an essential enzyme occurring in all biological kingdoms. In eukaryotes, it is located in the membrane of the endoplasmic reticulum. Selective inhibitors of trypanosomal CYP51s that do not affect the human CYP51 have been discovered in vitro and found to cure acute and chronic mouse Chagas disease without severe side effects in vivo. Crystal structures indicate that CYP51 may be more rigid than most CYPs, and it has been proposed that this property may facilitate antiparasitic drug design. Therefore, to investigate the dynamics of trypanosomal CYP51, we built a model of membrane‐bound Trypanosoma brucei CYP51 and then performed molecular dynamics simulations of T. brucei CYP51 in membrane‐bound and soluble forms. We compared the dynamics of T. brucei CYP51 with those of human CYP51, CYP2C9, and CYP2E1. In the simulations, the CYP51s display low mobility in the buried active site although overall mobility is similar in all the CYPs studied. The simulations suggest that in CYP51, pathway 2f serves as the major ligand access tunnel, and both pathways 2f (leading to membrane) and S (leading to solvent) can serve as ligand egress tunnels. Compared with the other CYPs, the residues at the entrance of the ligand access tunnels in CYP51 have higher mobility that may be necessary to facilitate the passage of its large sterol ligands. The water (W) tunnel is accessible to solvent during most of the simulations of CYP51, but its width is affected by the conformations of the heme's two propionate groups. These differ from those observed in the other CYPs studied because of differences in their hydrogen‐bonding network. Our simulations give insights into the dynamics of CYP51 that complement the available experimental data and have implications for drug design against CYP51 enzymes. Copyright © 2015 John Wiley & Sons, Ltd.     

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.     

Using Multiconformation Continuum Electrostatics to Compare Chloride Binding Motifs in α-Amylase, Human Serum Albumin, and Omp32

Ions are a ubiquitous component of the cellular environment, transferring into cells through membrane-embedded proteins. Ions bind to proteins to regulate their charge and function. Here, using multiconformation continuum electrostatics (MCCE), we show that the changes of chloride binding to α-amylase, human serum albumin (HSA) and Omp32 with pH, and of α-amylase with mutation agree well with experimental data. The three proteins represent three different types of binding. In α-amylase, chloride is bound in a specific buried site. Chloride binding is strongly coupled to the protonation state of a nearby lysine. MCCE calculates an 11-fold change in chloride affinity between the wild-type α-amylase and the K300R mutant, in good agreement with the measured 10-fold change. Without considering the coupled protonation reaction, the calculated affinity change would be more than 10⁶-fold. In HSA, chlorides are distributed on the protein surface. Although HSA has a negative net charge, it binds more anions than cations. There are no highly occupied binding sites in HSA. Rather, there are many partially occupied sites near clusters of basic residues. The relative affinity of bound ions of different charges is shown to depend on the distribution of charged residues on the surface rather than the overall net charge of the protein. The calculated strong pH dependence of the number of chlorides bound and the anion selectivity agree with those of previous experiments. In Omp32, chlorides are stabilized in an anion-selective transmembrane channel in a pH-independent manner. The positive electrostatic potential in Omp32 results in about two chlorides and no cations bound in the transmembrane region of this anion-selective channel. The studies here show that with the ability to sample multiple binding sites and coupled protein protonation states, MCCE provides a powerful tool to analyze and predict ion binding. The calculations overestimate the affinity of surface chloride in HSA and Omp32 relative to the buried ion in amylase. Differences between ion-solvent interactions for buried and surface ions will be discussed.     

Ammonia channeling in bacterial glucosamine-6-phosphate synthase (Glms): molecular dynamics simulations and kinetic studies of protein mutants

Ammonia transfer from the glutamine site to the fructose-6P site of bacterial glucosamine-6-phosphate synthase was studied by molecular dynamics simulations. The studies suggest a key role for Trp74, in the sealing of the hydrophobic channel connecting the two binding sites, as well as for the two Ala602 and Val605 residues, which form a narrow passage whose opening/closing constitutes an essential event in ammonia transfer. Kinetic analyses of the corresponding protein mutants confirmed our predictions. The efficiency of ammonia transfer which was close to zero in the W74A mutant was partially restored by increasing the size of the corresponding side-chain; the simulations performed on the W74A mutant suggested the formation of a hole in the channel. In the case of A602L and V605L mutants, the efficiency of ammonia transfer decreased to approximately 50% of the value of the native protein. None of the mutants were, however, able to use exogenous ammonia as a substrate.     

Molecular dynamics studies on mutants of Cu,Zn superoxide dismutase: The functional role of charged residues in the electrostatic loop VII

Molecular dynamics (MD) calculations have been performed on mutants of superoxide dismutase (SOD) on some residues present in the electrostatic loop. These calculations have provided the solution structures for the mutants Thr-137 → IIe and Arg; Lys-136 → Ala; Glu-132 → Gln; Glu-133 → Gln; Glu-132, Glu-133 → Gln-132, Gln-133 and → Gln-132, Lys-133. The structural and dynamic properties of these mutants have been correlated with the catalytic properties and available spectroscopic data. The water molecule present in the active site close to the copper ion in wild type (WT) SOD is missing in the MD average structure of the Thr-137 → IIe mutant, while this molecule is present in the MD average structures of all the other mutants and of WT SOD. This agrees with the experimental data. This is an important result that shows the validity of our calculations and their ability to reproduce even subtle structural features. Addition of one or more positive charges on the 132 and/or 133 positions does not sizably perturb the structure of the active site channel, while the introduction of a positively charged residue (Arg) on position 137 has a large effect on the structure of the electrostatic loop. Analysis of the MD average structures of these mutants has pointed out that the simple electrostatic effects of charged residues in the channel are not the only factor relevant for enzymatic behavior but that the structure of the electrostatic loop and the location of the charged residues also contribute to the catalytic properties of SOD. © 1994 John Wiley & Sons, Inc.     

Structural heterogeneity and ligand gating in ferric Methanosarcina acetivorans protoglobin mutants

Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, displays peculiar structural and functional properties within members of the hemoglobin superfamily. In fact, MaPgb-specific loops and a N-terminal extension (20 amino acid residues) completely bury the heme within the protein matrix. Therefore, the access of diatomic gaseous molecules to the heme is granted by two apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. The presence of two tunnels within the protein matrix could be partly responsible for the slightly biphasic ligand binding behavior. Unusually, MaPgb oxygenation is favored with respect to carbonylation. Here, the crucial role of Tyr(B10)61 and Ile(G11)149 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, on ligand binding to the heme-Fe-atom and on distal site structural organization is reported. In particular, tunnel 1 accessibility is modulated by a complex reorganization of the Trp(B9)60 and Phe(E11)93 side-chains, triggered by mutations of the Tyr(B10)61 and Ile(G11)149 residues, and affected by the presence and type of the distal heme-bound ligand.     

The first extracellular loop of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p undergoes a conformational change upon ligand binding

In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding.     

Structural determinants of ligand migration in Mycobacterium tuberculosis truncated hemoglobin O

Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, truncated hemoglobin N (trHbN) and truncated hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand-binding characteristics. The X-ray structure of trHbO has been solved and shows several internal cavities and secondary-docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer-simulation techniques. We have computed the free-energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple-ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand-migration regulation.     

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1–3 mutant

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1–3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Increased mobility in the membrane targeting PX domain induced by phosphatidylinositol 3-phosphate

Phosphoinositides (PIs) are concentrated in specific subcellular membranes in order to recruit and regulate cytosolic proteins responsible for vesicular trafficking, cytoskeletal rearrangement, and eukaryotic cell growth, differentiation, and survival. Phox homology (PX) domains are found in proteins that are integral players in endocytic pathways. For example, Vam7p is targeted by its PX domain to phosphatidylinositol 3-phosphate [PtdIns(3)P] in the yeast vacuole, where it interacts with other SNARE proteins and GTPases of the vesicular membrane fusion machinery. Although several PX structures have been solved, the role of dynamics in their interactions with membrane lipids is unclear. Here, we present the first detailed characterization of the backbone dynamics of a PX domain, that of Vam7p, in the presence and absence of its ligand. The structure appears to tumble more rapidly in solution upon binding PtdIns(3)P, revealing a conformational change that includes adjustments in the flexible membrane insertion loop (MIL). The flexibilities of the MIL and domain termini are pronounced in both states, while the alpha1 and alpha2 helices are rigid. Dynamic effects are spread across the binding pocket, with PtdIns(3)P inducing altered mobility of different residues on multiple timescales, including a shift in the MIL to slower timescale motions. The bound state is more dynamic overall, particularly in the beta-sheet lobe, which packs against the ligand's 3-phosphate. Thus, the induced dynamic and structural effects are transduced from the buried heart of the binding pocket in the helical lobe through the beta-sheet lobe to the exposed surface of the bilayer-inserted protein.     

Substrate tunnels in enzymes: Structure–function relationships and computational methodology

In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. Proteins 2015; 83:599–611. © 2015 Wiley Periodicals, Inc.     

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.     

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the alpha4'-helix-loop-alpha4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.     

How catalase recognizes H2O2 in a sea of water

Monofunctional heme‐catalases have been studied for many decades but there is still an incomplete understanding of why such a large tetrameric protein with deeply buried active sites is required to accomplish such a simple reaction as H₂O₂ dismutation. Catalase accomplishes this reaction at a high rate although water at 55 M is expected to compete with H₂O₂ for the enzyme's active site. Using molecular dynamics simulations we addressed the question as to how catalase selects H₂O₂ in water. Selection is accomplished through different mechanisms: higher residence time of H₂O₂ in the vicinity of certain prevalent amino acid residues at the protein surface and substrate channel, coordinated motion of the main passage amino acids that is increased in the presence of H₂O₂, a gate valve mechanism consisting of the motion of two contiguous phenylalanine residues that drive water molecules out of the final section of the substrate channel, a hydrophobic barrier before the active site that was crossed more easily by H₂O₂ which kept most of its hydrogen bonds while passing, and finally an increased residence time for H₂O₂ at the active site. These mechanisms, based on the physicochemical differences between H₂O₂ and water, provide an explanation as to why such a large tetrameric protein with deeply buried active sites is required to accomplish efficient H₂O₂ dismutation. Proteins 2014; 82:45–56. © 2013 Wiley Periodicals, Inc.     

Distal mutation modulates the heme sliding in mouse neuroglobin investigated by molecular dynamics simulation

Neuroglobin (Ngb), a hexa‐coordinated hemoprotein primarily expressed in the brain and retina, is thought to be involved in neuroprotection and signal transduction. Ngb can reversibly bind small ligands such as O₂ and CO to the heme iron by replacing the distal histidine which is bound to the iron as the endogenous ligand. In this work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamical characteristics in carboxy mouse neuroglobin and three distal mutants including single mutants H64V, K67T and double mutant H64V/K67T. MD simulations suggest that the heme sliding motion induced by the binding of exogenous ligand is affected by the distal mutation obviously. Accompanying changes in loop flexibility and internal cavities imply the structural rearrangement of Ngb. Moreover, the solvent accessibility of heme and some crucial residues are influenced revealing an interactive network on the distal side. The work elucidates that the key residues K67 at E10 and H64 at E7 are significant in modulating the heme sliding and hence the structural and physiological function of Ngb. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Insights into the role of d-amino acid oxidase mutations in amyotrophic lateral sclerosis

Missense mutations in the coding region of d -amino acid oxidase (DAO) have been found in patients suffering from amyotrophic lateral sclerosis (ALS). Mutations primarily impair the enzymatic activity of DAO and cause neurodegeneration due to an abnormal accumulation of d -serine in the spinal cord. However, the structural and dynamic changes that lead to impaired enzymatic activity are not fully understood. We present here extensive molecular dynamics simulations of wild-type, and all reported ALS-associated DAO mutants to elucidate the plausible mechanisms of impaired enzymatic activity, a critical function needed for neuroprotection. Simulation results show that DAO mutations disrupt several key interactions with the active site residues and decrease the conformational flexibility of active site loop comprising 216 to 228 residues, necessary for substrate binding and product release. This conformational restriction of the active site loop in the mutants is mainly due to the distortion of critical salt bridge and hydrogen bond interactions compared with wild-type. Furthermore, binding free energy calculations show that DAO mutants have a lower binding affinity toward cofactor flavin adenine dinucleotide and substrate imino-serine than the wild-type. A closer look at the cofactor and substrate interaction profiles further show that DAO mutants have lost several critical interactions with the neighboring residues as compared with wild-type. Taken together, this study provides first-hand explanation of crucial structural features that lead to the loss of enzymatic function in DAO mutants and highlights the need of further genomic scans of patients with ALS to map the association of novel DAO variants in ALS pathophysiology.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure–function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure–function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22–Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.