A dynamic view of enzyme catalysis

Recent experimental advances have shown that enzymes are flexible molecules, and point to a direct link between dynamics and catalysis. Movements span a wide time range, from nano- to milli-seconds. In this paper we introduce two aspects of enzyme flexibility that are treated with two appropriate techniques. First, transition path sampling is used to obtain an unbiased picture of the transition state ensemble in chorismate mutase, as well as its local flexibility and the energy flow during the chemical step. Second, we consider the binding and release of substrates in L-rhamnulose-1-phosphate aldolase. We have calculated the normal modes of the enzyme with the elastic network model. The lowest frequency modes generate active site deformations that change the coordination number of the catalytic zinc ion. The coordination lability of zinc allows the binding and release of substrates. Substitution of zinc by magnesium blocks the exchange of ligands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Thorough validation of protein normal mode analysis: a comparative study with essential dynamics

The deformation patterns of a large set of representative proteins determined by essential dynamics extracted from atomistic simulations and coarse-grained normal mode analysis are compared. Our analysis shows that the deformational space obtained with both approaches is quite similar when taking into account a representative number of modes. The results provide not only a comprehensive validation of the use of a low-frequency modal spectrum to describe protein flexibility, but also a complete picture of normal mode limitations.     

High pressure reveals that the stability of interdimeric contacts in the R- and T-state of HbA is influenced by allosteric effectors: Insights from computational simulations

The molecular details of the mechanism of action of allosteric effectors on hemoglobin oxygen affinity are not clearly understood. The global allostery model proposed by Yonetani et al. suggests that the binding of allosteric effectors can take place both in the R and T states and that they influence oxygen affinity through inducing global tertiary changes in the subunits. Recently published high pressure studies yielded dissociation constants at atmospheric pressure that showed a stabilizing effect of heterotropic allosteric effectors on the dimer interface in the R state, and a more pronounced destabilizing effect in a T state model. In the present work, we report on computational modeling used to interpret the high pressure experimental data. We show structural changes in the hemoglobin interdimeric interfaces, indicative of a global tertiary structural change induced by the binding of allosteric effectors. We also show that the number of water molecules bound at the interface is significantly influenced by binding effectors in the T state in accordance with the experimental data. Our results suggest that the binding of effectors at definite sites leads to tertiary changes that propagate to the interfaces and results in overall structural re-organizations.     

Molecular Dynamics of Class A β-lactamases—Effects of Substrate Binding

The effects of substrate binding on class A β-lactamase dynamics were studied using molecular dynamics simulations of two model enzymes; 40 100-ns trajectories of the free and substrate-bound forms of TEM-1 (with benzylpenicillin) and PSE-4 (with carbenicillin) were recorded (totaling 4.0 μs). Substrates were parameterized with the CHARMM General Force Field. In both enzymes, the Ω loop exhibits a marked flexibility increase upon substrate binding, supporting the hypothesis of substrate gating. However, specific interactions that are formed or broken in the Ω loop upon binding differ between the two enzymes: dynamics are conserved, but not specific interactions. Substrate binding also has a global structuring effect on TEM-1, but not on PSE-4. Changes in TEM-1’s normal modes show long-range effects of substrate binding on enzyme dynamics. Hydrogen bonds observed in the active site are mostly preserved upon substrate binding, and new, transient interactions are also formed. Agreement between NMR relaxation parameters and our theoretical results highlights the dynamic duality of class A β-lactamases: enzymes that are highly structured on the ps-ns timescale, with important flexibility on the μs-ms timescale in regions such as the Ω loop.     

Envisioning the Loop Movements and Rotation of the Two Subdomains of Dihydrofolate Reductase by Elastic Normal Mode Analysis

Abstract

Normal mode analysis, using the elastic network model, has been employed to envision the low frequency normal mode motion trends in the structures of five intermediates and a transition state in the kinetic pathway of E. coli dihydrofolate reductase (DHFR). Five of the reaction pathway analog structures and a crystal structure resembling the transition state, using X-ray analyses determined by Kraut et al., have been adapted as structural models. The motions that poise pathways of the M20 loop transitions from closed to occluded conformations and sub domain rotation to close the substrate cleft, have been predicted and envisioned for the first time by this study. Pathway entries to the movement of the substrate binding cleft helices are also envisioned. These motions play roles in transition structure stabilization and in regulating the release of the product tetrahydrofolate (THF). The motions observed push the ground state conformation of each intermediate towards a higher energy sub state conformation. A set of conserved residues involved in the catalytic reactions and conformational changes, previously studied by kinetic, theoretical and NMR, have been analyzed. The importance of these motions in terms of protein dynamics are revealed and envisioned by the normal mode analysis. Additional residues are proposed as candidates for further study of their potential promotional function.     

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca²⁺ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca²⁺ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein’s dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.     

Essential motions in a fungal lipase with bound substrate,covalently attached inhibitor and product

As an aid to understanding the influence of dynamic fluctuations during esterolytic catalysis, we follow protein flexibility at three different steps along the catalytic pathway from substrate binding to product clearance via a covalently attached inhibitor, which represents a transition-state mimic. We have applied a classical approach, using molecular dynamics simulations to monitor protein dynamics in the nanosecond regime. We filter out small amplitude fluctuations and focus on the anharmonic contributions to the overall dynamics. This 'essential dynamics' analysis reveals different modes of response along the pathway suggesting that binding, catalysis and product clearance occur along different energy surfaces. Motions in the enzyme with a covalently attached ligand are more complex and occur along several eigenvectors. The magnitudes of the fluctuations in these individual subspaces are significantly smaller than those observed for the substrate and product molecules, indicating that the energy surface is shallow and that a relatively large number of conformational substates are accessible. On the other hand, substrate binding and product release occur at distinct modes of the protein flexibility suggesting that these processes occur along rough energy surfaces with only a few minima. Detailed energetic analyses along the trajectories indicated that in all cases binding is dominated by van der Waals interactions. The carboxylate form of the product is stabilized by a tight hydrogen bond network involving in particular Ser82, which may be a potential cause of product inhibition. Considerations such as these should aid the understanding of mechanisms of substrate, inhibitor or product recognition and could become of importance in the design of new substrates or inhibitors for enzymes.     

Enzyme specificity under dynamic control II: Principal component analysis of α-lytic protease using global and local solvent boundary conditions

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190-->Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. Based on a combination of multiconformation analysis of cryo-X-ray crystallographic data, solution nuclear magnetic resonance (NMR), and normal mode calculations, we had hypothesized that the large alteration in specificity of the mutant enzyme is mainly attributable to changes in the dynamic movement of the two walls of the specificity pocket. To test this hypothesis, we performed a principal component analysis using 1-nanosecond molecular dynamics simulations using either a global or local solvent boundary condition. The results of this analysis strongly support our hypothesis and verify the results previously obtained by in vacuo normal mode analysis. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity. As shown here, concerted local movements within proteins can be efficiently analyzed through a combination of principal component analysis and molecular dynamics trajectories using a local solvent boundary condition to reduce computational time and matrix size.     

On the Conservation of the Slow Conformational Dynamics within the Amino Acid Kinase Family: NAGK the Paradigm

N-Acetyl-L-Glutamate Kinase (NAGK) is the structural paradigm for examining the catalytic mechanisms and dynamics of amino acid kinase family members. Given that the slow conformational dynamics of the NAGK (at the microseconds time scale or slower) may be rate-limiting, it is of importance to assess the mechanisms of the most cooperative modes of motion intrinsically accessible to this enzyme. Here, we present the results from normal mode analysis using an elastic network model representation, which shows that the conformational mechanisms for substrate binding by NAGK strongly correlate with the intrinsic dynamics of the enzyme in the unbound form. We further analyzed the potential mechanisms of allosteric signalling within NAGK using a Markov model for network communication. Comparative analysis of the dynamics of family members strongly suggests that the low-frequency modes of motion and the associated intramolecular couplings that establish signal transduction are highly conserved among family members, in support of the paradigm sequence→structure→dynamics→function.     

Instantaneous Normal Modes and the Protein Glass Transition

In the instantaneous normal mode method, normal mode analysis is performed at instantaneous configurations of a condensed-phase system, leading to modes with negative eigenvalues. These negative modes provide a means of characterizing local anharmonicities of the potential energy surface. Here, we apply instantaneous normal mode to analyze temperature-dependent diffusive dynamics in molecular dynamics simulations of a small protein (a scorpion toxin). Those characteristics of the negative modes are determined that correlate with the dynamical (or glass) transition behavior of the protein, as manifested as an increase in the gradient with T of the average atomic mean-square displacement at ∼220 K. The number of negative eigenvalues shows no transition with temperature. Further, although filtering the negative modes to retain only those with eigenvectors corresponding to double-well potentials does reveal a transition in the hydration water, again, no transition in the protein is seen. However, additional filtering of the protein double-well modes, so as to retain only those that, on energy minimization, escape to different regions of configurational space, finally leads to clear protein dynamical transition behavior. Partial minimization of instantaneous configurations is also found to remove nondiffusive imaginary modes. In summary, examination of the form of negative instantaneous normal modes is shown to furnish a physical picture of local diffusive dynamics accompanying the protein glass transition.     

HIV-1跨膜蛋白gp41与N-取代吡咯衍生物的结合模式研究

采用分子对接,分子动力学(MD)模拟和分子力学/泊松-波尔兹曼溶剂可有面积方法与分子力学/广义伯恩溶剂可及面积方法(MM-PBSA/MM-GBSA),预测两种N-取代吡咯衍生物与HIV-1 跨膜蛋白gp41疏水口袋的结合模式与作用机理．分子对接采用多种受体构象,并从结果中选取几种可能的结合模式进行MD 模拟,然后通过MM-PBSA计算结合能的方法识别最优的结合模式. MM-PBSA计算结果表明,范德华相互作用是结合的主要驱动力,而极性相互作用决定了配体在结合过程中的取向．进一步的结合能分解显示,配体的羧基与gp41残基Arg579的静电相互作用对结合有重要贡献．上述工作为进一步优化N-取代吡咯衍生物类的HIV-1融合抑制剂建立了良好的理论基础．     

Group theory of icosahedral virus capsid vibrations: A top-down approach

We explore the use of a top-down approach to analyse the dynamics of icosahedral virus capsids and complement the information obtained from bottom-up studies of viral vibrations available in the literature. A normal mode analysis based on protein association energies is used to study the frequency spectrum, in which we reveal a universal plateau of low-frequency modes shared by a large class of Caspar-Klug capsids. These modes break icosahedral symmetry and are potentially relevant to the genome release mechanism. We comment on the role of viral tiling theory in such dynamical considerations.     

Prediction of the binding mode between BMS-378806 and HIV-1 gp120 by docking and molecular dynamics simulation

BMS-378806 is a newly discovered small molecule that effectively blocks the binding of CD4 with gp120. The binding mode of this kind of inhibitor remains unknown. In this paper, AutoDock 3.0 in conjunction with molecular dynamics simulation, accommodating the receptor's flexibility, was used to explore the binding mode between BMS-378806 and gp120. Two structures, Mode I and Mode II, with the lowest docking energy were selected as different representative binding modes. The analysis of the results from the molecular dynamics simulation indicated that the binding of BMS-348806 in Mode II is more stable. The average structure of Mode II was analyzed and compared with the experimental data. The conclusion was that BMS-378806 inserts the azaindole ring deeply into the PHE43 cavity and makes contact with a number of residues in the cavity, on the cavity and near the cavity. This study benefits the understanding of the mechanism of this kind of inhibitor and may provide useful information for rational drug design.     

Molecular modeling studies of substrate binding by penicillin acylase

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.     

Transconformations of the SERCA1 Ca-ATPase: a normal mode study

The transport of Ca(2+) by Ca-ATPase across the sarcoplasmic reticulum membrane is accompanied by several transconformations of the protein. Relying on the already established functional importance of low-frequency modes in dynamics of proteins, we report here a normal mode analysis of the Ca(2+)-ATPase based on the crystallographic structures of the E1Ca(2) and E2TG forms. The lowest-frequency modes reveal that the N and A(+Nter) domains undergo the largest amplitude movements. The dynamical domain analysis performed with the DomainFinder program suggests that they behave as rigid bodies, unlike the highly flexible P domain. We highlight two types of movements of the transmembrane helices: i), a concerted movement around an axis perpendicular to the membrane which "twists open" the lumenal side of the protein and ii), an individual translational and rotational mobility which is of lower amplitude for the helices hosting the calcium binding sites. Among all modes calculated for E1Ca, only three are enough to describe the transition to E2TG; the associated movements involve almost exclusively the A and N domains, reflecting the closure of the cytoplasmic headpiece and high displacement of the L7-8 lumenal loop. Subsequently, we discuss the potential contribution of the remaining low-frequency normal modes to the transconformations occurring within the overall calcium transport cycle.