Computational analysis of chain flexibility and fluctuations in Rhizomucor miehei lipase

We have performed molecular dynamics simulation of Rhizomucormiehei lipase (Rml) with explicit water molecules present. Thesimulation was carried out in periodic boundary conditions andconducted for 1.2 ns in order to determine the concerted proteindynamics and to examine how well the essential motions are preservedalong the trajectory. Protein motions are extracted by meansof the essential dynamics analysis method for different lengthsof the trajectory. Motions described by eigenvector 1 convergeafter approximately 200 ps and only small changes are observedwith increasing simulation time. Protein dynamics along eigenvectorswith larger indices, however, change with simulation time andgenerally, with increasing eigenvector index, longer simulationtimes are required for observing similar protein motions (alonga particular eigenvector). Several regions in the protein showrelatively large fluctuations and in particular motions in theactive site lid and the segments Thr57–Asn63 and the activesite hinge region Pro101–Gly104 are seen along severaleigenvectors. These motions are generally associated with glycineresidues, while no direct correlations are observed betweenthese fluctuations and the positioning of prolines in the proteinstructure. The partial opening/closing of the lid is an exampleof induced fit mechanisms seen in other enzymes and could bea general mechanism for the activation of Rml.     

Concerted motions in the photoactive yellow protein

Molecular dynamics simulations have been performed with the aim of identifying concerted backbone motions in the photoactive yellow protein. Application of the essential dynamics method revealed large, chromophore-linked fluctuations of the protein in the ground state, as well as in a form containing the isomerized chromophore. Various loops become more mobile upon isomerization of the chromophore, including a loop which is part of the PAS domain motif, found in light perception proteins. The hinge points identified in these fluctuations correlate with the positions of evolutionary conserved glycines. The results derived from the simulations directly correlate with available experimental data, provide a framework for understanding the dynamic behaviour of the yellow protein and give clues to subsequent steps in the signal transduction pathway.      

Protein dynamics determined by backbone conformation and atom packing

To study the factors determining the collective motions in thermal, conformational fluctuations of a globular protein, molecular dynamics simulations were performed with a backbone model and an atomic-level model. In the backbone model, only the C alpha atoms were explicitly treated with two types of pairwise interactions assigned between the C alpha atoms; atom-packing interactions to take into account the effect of tight atom packing in the protein interior and chain-restoring interactions to maintain the backbone around the native conformation. A quasi-harmonic method was used to decompose the overall fluctuations into independent, collective modes. The modes assigned to large conformational fluctuations showed a good correlation between the backbone and atomic-level models. From this study, it was suggested that the collective modes were motions in which a protein fluctuates, keeping the tertiary structure around the native one and avoiding backbone overlap and, hence, rough aspects of the collective modes can be derived without details of the atomic interactions. The backbone model is useful in obtaining the overall backbone motions of a protein without heavy simulations, even though the simulation starts from a poorly determined conformation of experiments and in sampling main chain conformations, from which the side chain conformations may be predicted.      

Molecular dynamics of acetylcholinesterase

Molecular dynamics simulations are leading to a deeper understanding of the activity of the enzyme acetylcholinesterase. Simulations have shown how breathing motions in the enzyme facilitate the displacement of substrate from the surface of the enzyme to the buried active site. The most recent work points to the complex and spatially extensive nature of such motions and suggests possible modes of regulation of the activity of the enzyme.     

Characterization of enzyme motions by solution NMR relaxation dispersion

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo or off-resonance R 1rho pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Deltaomega) between the conformations. The CPMG experiments were applied to the backbone N-H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme-substrate (ES) complex, and enzyme-product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s (-1) and is the same as both k cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k ex derived from the NMR measurements and k cat from enzyme kinetic studies. This isotope effect of approximately 2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function. For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R 1rho experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10,000 s (-1), which is the same as the catalytic rate constant. The O (eta) atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in the equilibrium population of the open conformer of loop 6, thereby resulting in a loss of activity through a shift in the conformational equilibrium of loop 6. Solution NMR relaxation dispersion experiments are powerful experimental tools that can elucidate protein motions with atomic resolution and can provide insight into the role of these motions in biological function.     

Fatty acid desaturase systems of hen liver and their inhibition by cyclopropene fatty acids

Hen liver preparations which desaturate stearic acid at the 9,10 position to form oleic acid have been found to desaturate other saturated fatty acids of carbon chain length from 12 to 20 and 22. The 9,10-monoenoic fatty acid of the same chain length as the substrate fatty acid is the major product formed. Minor amounts of the 10,11- and 11, 12-monoenoic acids are also formed. Maximum desaturation occurred with the C₁₄ fatty acid substrate and with the fatty acids C₁₇ and C₁₈, suggesting the presence of at least two desaturating systems. The cyclopropene fatty acids, sterculic and malvalic acids, inhibited the desaturation of all thefatty acids at the 9,10 position but desaturation at the 10,11 and 11, 12 positions was affected only slightly. The effect is not due to inhibition of the primary activating enzyme, the long chain acyl CoA synthetase. Sterculic acid is a more effective inhibitor than either malvalic acid or sterculyl alcohol, probably because these cyclopropene compounds do not block the desaturating site of the enzyme as completely as sterculic acid.     

Modulating functional loop movements: the role of highly conserved residues in the correlated loop motions

Loop flexibility in enzymes plays a vital role in correctly positioning catalytically important residues. This strong relationship between enzyme flexibility and function provides an opportunity to engineer new substrates and inhibitors. It further allows the design of site-directed mutagenesis experiments to explore enzymatic activity through the control of flexibility of a functional loop. Earlier, we described a novel mechanism in which a small loop triggers the motions of a functional loop in three enzymes (beta-1,4-galactosyltransferase, lipase, and enolase) unrelated in sequence, structure, or function. Here, we further address the question of how the interactions between various flexible loops modulate the movements of the functional loop. We examine beta-1,4-galactosyltransferase as a model system in which a Long loop undergoes a large conformational change (moves in space up to 20 A) upon substrate binding in addition to a small loop (Trp loop) that shows a considerably smaller conformational change. Our molecular-dynamics simulations carried out in implicit and explicit solvent show that, in addition to these two loops, two other neighboring loops are also highly flexible. These loops are in contact with either the Long loop or the Trp loop. Analysis of the covariance of the spatial displacement of the residues reveals that coupled motions occur only in one of these two loops. Sequence analysis indicates that loops correlated in their motions also have highly conserved residues involved in the loop-loop interactions. Further, analysis of crystal structures and simulations in explicit water open the possibility that the Trp loop that triggers the movement of the Long loop in the unbound conformation may also play the same role in the substrate-bound conformation through its contact with the conserved and correlated third loop. Our proposition is supported by the observation that four of the five conserved positions in the third loop are at the interface with the Trp loop. Evolution appears to select residues that drive the functional Long loop to a large conformational change. These observations suggest that altering selected loop-loop interactions might modulate the movements of the functional loop.     

Molecular basis of lipase stereoselectivity

Lipases exhibit specific catalytic properties that make them attractive to biotechnological applications. Most important are the broad substrate specificity and the regio‐ and stereoselectivity of lipases. Despite mechanistic and structural similarities lipases differ significantly with respect to stereoselectivity toward natural and synthetic substrates. Models developed to describe and predict stereoselectivity toward certain types of synthetic substrates, e. g., secondary alcohols cannot be applied to natural acylglycerols, that are hydrolyzed by several animal and microbial lipases in a regioselective or stereoselective manner. Therefore, computer‐aided molecular modeling studies were used in order to predict the stereopreference of lipases toward triradylglycerols. Lipase variants with modified stereoselectivity properties toward triacylglycerols were engineered by re‐designing the recombinant enzyme. To understand the interactions governing lipase stereoselectivity towards natural substrates, knowledge of the structure of enzyme‐substrate complexes at the atomic level is essential. Such information can be obtained by X‐ray or NMR analysis of covalent enzyme‐inhibitor complexes. The crystal structures of enzymes complexed with triacylglycerol analog inhibitors allowed the identification of distinct binding sites for the three hydrophobic chains of the inhibitor.     

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

Annexins are homologous proteins that bind to membranes in a calcium dependent manner, but for which precise physiological roles have yet to be defined. Most annexins are composed of a planar array of four homologous repeats, each containing five alpha-helices and associated into two modules. Annexin V forms a voltage-gated calcium channel in phospholipid bilayers. It has been proposed that the hydrophilic pore in the centre of the molecule may represent the ion conduction pathway and that a hinge movement in annexin V causes a variation of the inter- module angle and opens the calcium ion path. Here we present the results of molecular dynamics simulations of apo-annexin V and of calcium-bound annexin V and annexin I. The three simulations show significant differences in conformation and dynamics. The essential dynamics method was used to study the essential subspace of annexin V and showed that one of the essential motions corresponds to the postulated hinge motion. The hinge residues were located between repeats but belong to helices rather than to the links between helices. Calcium binding to annexin V led to a limitation of this hinge motion with more open conformations being favoured.      

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.     

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

A theoretical study of substrate-induced activation of dienelactone hydrolase

Dienelactone hydrolase (DLH), an enzyme from the ß-ketoadipatepathway, catalyses the hydrolysis of dienelactone to maleylacetate.DLH is unusual because it is the only known naturally occurringenzyme which contains the catalytic triad Cys...His...Asp. Thistriad has previously been created artificially in the mutantserine proteases, thiol subtilisin and thiol trypsin. In bothcases the mutant enzymes exhibited activities several ordersof magnitude lower than the wild type enzymes; the low reactivityhas generally been attributed to the inability of these enzymesto form a catalytically active thiolate anion (Cys^– ...His⁺...Asp^–).The crystal structure of DLH suggests that the native enzymeexists predominantly in a catalytically inert configuration;the triad cysteine is neutral and points away from the activesite binding cleft. However, a crystallographic analysis ofC123S DLH complexed with an isostructural inhibitor (dienelactam)indicates that substrate binding induces a prototropic rearrangementof the active site prior to catalysis which results in the formationof a highly nucleophilic thiolate anion. We have performed abinitio SCF/MP2 calculations on a relatively small portion ofthe active site of DLH to examine the details of this activationprocess. Our calculations provide supporting evidence that theconformational changes observed in the crystal structure dueto inhibitor (or substrate) binding facilitate the formationof a reactive thiolate anion. In particular, substrate bindingalters the position of Glu36; the carboxylate side chain ofGlu36 is pushed towards C123 enabling it to abstract the thiolproton thus creating a catalytically active thiolate anion.The calculations also provide a possible explanation for thelow reactivities observed in the mutant serine proteases.     

Characterization of a chemically modified β-amylase immobilized on porous silica

β-Amylase was coupled to a periodate oxidized dextran by reductive alkylation in the presence of sodium cyanoborohydride. The loss of activity (57%) during the cross-linking of the enzyme was the result of steric hindrance near the catalytic site. In order to verify this hypothesis, the residual activity was determined with substrates of variable molecular size. The residual activity was inversely proportional to the particulate size of the substrate. Increases in residual activity, of up to 53% were obtained using an orientated chemical modification in the presence of a substrate which protects the catalytic site. Native and dextran-conjugated β-amylase were immobilized on an amino activated silica by a classical method using glutaraldehyde for the native enzyme and by reductive alkylation for the modified enzyme. The relative activity of the enzymes obtained after this insolubilization was very high for the modified amylase, 45% for a medium enzyme concentration, compared with 4% at the same concentration for the native enzyme. This difference can be attributed to the variation of the length of the space arm between the silica and the enzyme. The soluble β-amylase dextran conjugate had a superior thermoresistance to that of the native enzyme; its optimal temperature of activity was 5°C higher than that of the native enzyme. This stabilization may be attributable to a rigidification of the protein structure. Immobilization of both native and modified enzymes on a amino silica resulted in thermostabilization of the enzymes. The optimal temperature of activity was 70°C for the native immobilized β-amylase (some 10°C higher than that of the native enzyme) and 75°C for the chemically modified, and subsequently immobilized, β-amylase. The immobilized forms of the enzymes were used for 14 days at 55°C in continuous substrate processing. The greater eflciency of the chemically modified β-amylase was demonstrated by a four-fold increase in maltose production compared to the classical method of immobilization. A kinetic study confirmed the stabilization of the β-amylase by a reduction of the rate constant of inactivation of the different modified enzymes.     

A salt-bridge controlled by ligand binding modulates the hydrolysis reaction in a GH5 endoglucanase

Cellulases, distributed in at least 15 families of glycoside hydrolases, will play a key role in biomass conversion and renewable energy challenges of the future. Cel5B from Clostridium thermocellum is a β-1,4-endoglucanase and a member of family 5 of glycoside hydrolases (GH5) and is characterized by an (α/β)(8) barrel structure. In contrast to other retaining enzymes, in which the catalytic carboxylate groups (glutamate or aspartate) are positioned ≈ 5.5 ? apart to facilitate nucleophilic attack on the anomeric carbon of the sugar substrate, these two residues in Cel5B are positioned ≈ 10 ? from each other in the unliganded wild-type structure. The structure of the enzyme solved in complex with a cleavage product (cellobiose) revealed ligand-induced conformational changes in the loop carrying Glu140 (proton donor). The reorientation of Glu140 in the complex reduces the separation of the catalytic glutamate residues to 4.3 ?. In this study, we took advantage of conventional and steered molecular dynamics (MD) simulations along with in silico and in vitro mutagenesis to investigate the ligand-induced changes of the enzyme and interactions involved in preservation of Cel5B conformations in the presence and absence of substrate. We determined that the variation in separation of catalytic glutamates in the absence and presence of substrate is due to the different protonation states of the proton donor glutamate that is largely governed by conformational changes in the β3α3 loop. In the absence of substrate, the conformation of Cel5B is preserved by an electrostatic interaction between deprotonated Glu140 and protonated His91. The ion pair is interrupted upon the binding of substrate, and the positional displacement of the β3α3 loop allows Glu140 to become oriented within the active site in a less hydrophilic microenvironment that assists in Glu140 protonation.     

Understanding the Key Factors that Control the Inhibition of Type II Dehydroquinase by (2R)‐2‐Benzyl‐3‐dehydroquinic Acids

The binding mode of several substrate analogues, (2R)‐2‐benzyl‐3‐dehydroquinic acids 4 , which are potent reversible competitive inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway, has been investigated by structural and computational studies. The crystal structures of Mycobacterium tuberculosis and Helicobacter pylori DHQ2 in complex with one of the most potent inhibitor, p‐methoxybenzyl derivative 4 a , have been solved at 2.40 Å and 2.75 Å, respectively. This has allowed the resolution of the M. tuberculosis DHQ2 loop containing residues 20–25 for the first time. These structures show the key interactions of the aromatic ring in the active site of both enzymes and additionally reveal an important change in the conformation and flexibility of the loop that closes over substrate binding. The loop conformation and the binding mode of compounds 4 b – d has been also studied by molecular dynamics simulations, which suggest that the benzyl group of inhibitors 4 prevent appropriate orientation of the catalytic tyrosine of the loop for proton abstraction and disrupts its basicity.     

Macromolecular motions and hydrodynamic radius variation in dilute solutions under shear action

Understanding the dynamics of single polymer chains and rheological mechanism in dilute polymer solutions under shear stress is essential for fields such as the petroleum and food industries, biomedical materials and drug delivery. Here we present an experimental method for measuring the viscosity of polymer solutions and studying the variation of single polymer chain conformation and the mechanism of molecular motions according to the relationship between the intrinsic viscosity, [η], and the shear rate. Of striking interest is that we find that [η] changing with the shear rate presents three stages which may explain the nature of the viscoelastic performance of polymer solutions and the isolated molecular motions. The significance of these results is the finding of the polymer chain deformation to match the pore throat which has enormous potential implications in drug delivery, genetics and biomedicine © 2014 Society of Chemical Industry.     

A Second,Druggable Binding Site in UDP‐Galactopyranose Mutase from Mycobacterium tuberculosis?

UDP‐galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non‐substrate‐like inhibitor, MS‐208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS‐208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS‐208 binds at an allosteric binding site (A‐site) instead of the enzyme active site (S‐site). A candidate A‐site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A‐site. Further molecular dynamics studies led us to propose that MS‐208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A‐site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.     

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residueAsn14 that plays a crucial role in stabilizing the reactivesite loop conformation. This residue is found to be conservedin the Kunitz (STI) family of serine protease inhibitors. Tounderstand the contribution of this scaffolding residue on thestability of the reactive site loop, it was mutated in silicoto Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD)simulations were carried out on the mutants. The results ofMD simulations reveal the conformational variability and rangeof motions possible for the reactive site loop of differentmutants. The N-terminus side of the scissile bond, which isclose to a ß-barrel, is conformationally less variable,while the C-terminus side, which is relatively far from anysuch secondary structural element, is more variable and needsstability through hydrogen-bonding interactions. The simulatedstructures of WCI and the mutants were docked in the peptide-bindinggroove of the cognate enzyme chymotrypsin and the ability toform standard hydrogen-bonding interactions at P3, P1 and P2'residues were compared. The results of the MD simulations coupledwith docking studies indicate that hydrophobic residues likeLeu and Val at the 14th position are disruptive for the integrityof the reactive site loop, whereas a residue like Thr, whichcan stabilize the C-terminus side of the scissile bond, canbe predicted at this position. However, the size and chargeof the Asn residue made it most suitable for the best maintenanceof the integrity of the reactive site loop, explaining its conservednature in the family. Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.     

Bifunctional Binding and Catalysis in Host-Guest Chemistry

Some dimers of cyclodextrin have been prepared that can bind appropriate substrates in water with binding constants as strong as those of strong antigen/antibody complexes. Doubly linking two cyclodextrins with connections at neighboring glucose units produces a hinged molecule that resembles a clamshell. Another isomer is also produced, in the so-called loveseat configuration, in which the two cyclodextrin units cannot cooperate in binding a single substrate. When the two links are of different length, the clamshell dimer preferentially binds a non-linear substrate with an affinity exceeding 10¹¹ M⁻¹, at least 10³ better than the binding of a linear substrate isomer. Binding of substrates occurs by induced fit, in which the host closes around and engulfs the substrate. Mechanistic studies on models for the enzyme ribonuclease have led to a proposed mechanism different from that normally suggested for the enzyme. This mechanism has guided the synthesis of a new enzyme mimic with improved properties. Isotope studies show that this enzyme mimic uses a simultaneous bifunctional mechanism, as does the enzyme. The double binding of the transition state by the two imidazole units of the enzyme mimic occurs because of their linkage. The geometric preference for this binding reflects anon-linear transition state.