Molecular Mechanics Evaluation of the Proposed Mechanisms for the Degradation of Urea by Urease

Abstract

A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the N_His272-Ni1-O-C_urea and Ni1-O-Curea-N_urea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional “His320 acts as a base” mechanism is unlikely, while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.     

A molecular mechanical analysis of the active site of urease with a special emphasis on determining the binding conformations available to oxygen-bound urea.

In order to model the active site of urease which contains two nickel ions with differing coordination geometries new parameters were derived for the AMBER* force field. These parameters were obtained by structure based optimization and use a single set of parameters with points on a sphere approach to model nickel(II) high-spin in all its coordination geometries. The force field was successfully used to model the active site of urease and to predict that a bridging water between the two nickel ions in urease was missing from the solid state structure of urease. A thorough conformational search was undertaken to find the conformations available to urea within urease. All the low energy conformations found were used to determine a consensus urea binding model.     

Catalyzed decomposition of urea. Molecular dynamics simulations of the binding of urea to urease

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0 A crystal structure of Benini et al. [(1999) Struct. Folding Des. 7, 205-216] of DAP-inhibited urease (PDB code ), and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. [(1991) J. Am. Chem. Soc. 113, 8262-8270]. Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time-averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A "protein-assisted proton transfer" mechanism points to Asp221 as crucial for catalysis. An "Asp-mediated proton transfer" involves the transfer of a proton from the bridging OH to an NH2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis is discussed in detail.     

New stochastic strategy to analyze helix folding

We propose an alternative stochastic strategy to search secondary structures based on the generalized simulated annealing (GSA) algorithm, by using conformational preferences based on the Ramachandran map. We optimize the search for polypeptide conformational space and apply to peptides considered to be good alpha-helix promoters above a critical number of residues. Our strategy to obtain conformational energies consist in coupling a classical force field (THOR package) with the GSA procedure, biasing the Phi x Psi backbone angles to the allowed regions in the Ramachandran map. For polyalanines we obtained stable alpha-helix structures when the number of residues were equal or exceeded 13 amino acids residues. We also observed that the energy gap between the global minimum and the first local minimum tends to increase with the polypeptide size. These conformations were generated by performing 2880 stochastic molecular optimizations with a continuum medium approach. When compared with molecular dynamics or Monte Carlo methods, GSA can be considered the fastest.     

Evolutionary-inspired probabilistic search for enhancing sampling of local minima in the protein energy surface

Background

Despite computational challenges, elucidating conformations that a protein system assumes under physiologic conditions for the purpose of biological activity is a central problem in computational structural biology. While these conformations are associated with low energies in the energy surface that underlies the protein conformational space, few existing conformational search algorithms focus on explicitly sampling low-energy local minima in the protein energy surface.

Methods

This work proposes a novel probabilistic search framework, PLOW, that explicitly samples low-energy local minima in the protein energy surface. The framework combines algorithmic ingredients from evolutionary computation and computational structural biology to effectively explore the subspace of local minima. A greedy local search maps a conformation sampled in conformational space to a nearby local minimum. A perturbation move jumps out of a local minimum to obtain a new starting conformation for the greedy local search. The process repeats in an iterative fashion, resulting in a trajectory-based exploration of the subspace of local minima.

Results and conclusions

The analysis of PLOW's performance shows that, by navigating only the subspace of local minima, PLOW is able to sample conformations near a protein's native structure, either more effectively or as well as state-of-the-art methods that focus on reproducing the native structure for a protein system. Analysis of the actual subspace of local minima shows that PLOW samples this subspace more effectively that a naive sampling approach. Additional theoretical analysis reveals that the perturbation function employed by PLOW is key to its ability to sample a diverse set of low-energy conformations. This analysis also suggests directions for further research and novel applications for the proposed framework.

     

Conformational Analysis of Tetracycline using Molecular Mechanical and Semiempirical MO-Calculations

A combination of gas phase molecular dynamics and systematic conformational searches with semiempirical MO-theory has been used to identify and characterise the low energy conformations of tetracycline in its neutral non-polar and zwitterionic forms and as the deprotonated zwitteranion. The conformations found have been characterised by cluster- and principal components analyses as belonging to three main groups. These are very similar for the nonpolar and zwitterionic neutral forms of tetracycline, but very different for the anion. The likely consequences of the conformational flexibilty for metal ion complexation by tetracycline are discussed.Electronic Supplementary Material available.     

Optimal protocol and trajectory visualization for conformational searches of peptides and proteins.

Conformational searches by molecular dynamics and different types of Monte Carlo or build-up methods usually aim to find the lowest-energy conformation. However, this is often misleading, as the energy functions used in conformational calculations are imprecise. For instance, though positions of local minima defined by the repulsive part of the Lennard-Jones potential are usually altered only slightly by functional modification, the relative depths of the minima could change significantly. Thus, the purpose of conformational searches and, correspondingly, performance criteria should be reformulated and appropriate methods found to extract different local minima from the search trajectory and allow visualization in the search space. Attempts at convergence to the lowest-energy structure should be replaced with efforts to visit a maximum number of different local energy minima with energies within a certain range. We use this quantitative criterion consistently to evaluate performances of different search procedures. To utilize information generated in the course of simulation, a "stack" of low energy conformations is created and stored. It keeps track of variables and visit numbers for the best representatives of different conformational families. To visualize the search, projection of multidimensional walks onto a principal plane defined by a set of reference structures is used. With Met-enkephalin as a structural example and a Monte Carlo procedure combined with energy minimization (MCM) as a basic search method, we analyzed the influence on search efficiency of different characteristics as temperature schedules, the step size for variable modification, constrained random step and response mechanisms to search difficulties. Simulated annealing MCM had comparable efficiency with MCM at constant and elevated temperature (about 600 K). Constraining the randomized choice of side-chain chi angles to optimal values (rotamers) on every MCM step did not improve, but rather worsened, the search efficiency. Two low-energy Met-enkephalin conformations with parallel Tyr1 and Phe4 rings, a gamma-turn around the Gly2 residue, and Phe4 and Met5 side-chains forming together a compact hydrophobic cluster were found and are suggested as possible structural candidates for interaction with a receptor or a membrane.     

Systematic conformational investigations of peptoids and peptoid-peptide chimeras

Peptoids are originally defined as N-substituted oligoglycine derivatives, and in a broader definition as N-substituted peptides (peptoid-peptide chimeras). Both types were systematically investigated by force field calculations. The Merck MMFF and YASARA2 force fields were shown to be, among others, the most suitable ones for conformational investigations of peptoids with no missing parameterizations, in contrast to AMBER or CHARMM. Ramachandran-like plots were calculated for dipeptoids and chimeras using energy calculations and grid searches by varying the dihedral angels PHI and PSI in steps of 10 degrees for s-cis- and s-trans amide bonds. Barriers as well as low energy conformations are compared to peptide Ramachandran plots, showing that peptoids have both, more barriers due to additional steric interactions as well as access to minimum conformations not accessible by peptides. Low energy conformations of dimers were used as starting conformations of higher oligomers of the peptoids for extensive molecular dynamics simulations over 10 or 20 ns with the YASARA2 force field and an explicit water solvent box to evaluate their potential to form secondary structural elements. Especially peptoids with aminoisobutyric acid-like monomer units were found to form left-handed or polyproline-like helices also known from less common natural peptides. Furthermore, new secondary structures appear feasible based on stable conformations outside the allowed areas of the Ramachandran plot for peptides, but allowed for peptoids.     

Stereochemical studies on cyclic peptides: Detailed energy minimization studies on hydrogen bonded all-trans cyclic pentapeptide backbones

Conformational studies have been carried out on hydrogenbonded all-trans cyclic pentapeptide backbone. Application of a combination of grid search and energy minimization on this system has resulted in obtaining 23 minimum energy conformations, which are characterized by unique patterns of hydrogen bonding comprising of β- and γ-turns. A study of the minimum energy conformationsvis-a-vis non-planar deviation of the peptide units reveals that non-planarity is an inherent feature in many cases. A study on conformational clustering of minimum energy conformations shows that the minimum energy conformations fall into 6 distinct conformational families. Preliminary comparison with available X-ray structures of cyclic pentapeptide indicates that only some of the minimum energy conformations have formed crystal structures. The set of minimum energy conformations worked out in the present study can form a consolidated database of prototypes for hydrogen bonded backbone and be useful for modelling cyclic pentapeptides both synthetic and bioactive in nature. This is part XV of the series. Part XIV in this series is Ramakrishnanet al 1987.     

Structure-based computational study of the catalytic and inhibition mechanisms of urease

The viability of different mechanisms of catalysis and inhibition of the nickel-containing enzyme urease was explored using the available high-resolution structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates. The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calculated using ab initio and density functional theory methods. The DOCK program suite was employed to determine families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria. Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calculation of best energy structures for the nickel-bound inhibitors beta-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid. These calculated structures are in good agreement with the experimentally determined structures, and provide hints on the reactivity and mobility of the inhibitors in the active site. The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site. These calculations suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea molecule, with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.     

Computer simulations of cyclic enkephalin analogues

Molecular dynamics simulations and energy minimization studies of cyclic enkephalin analogues incorporating retro-inverso modifications have been carried out. The dynamic trajectories are analyzed in terms of the relative mobility of the 14-membered rings, conformational transitions among equilibrium states, and hydrogen-bonding patterns. The cyclization of the molecules reduces the motion of the ring structures substantially. Time-correlated conformational transitions resulting in the reorientation of peptide units are observed. Hydrogen bonds form principally C7 structures. Because of the incorporation of retro-inverso residues, C6 and C8 structures are also formed. Starting conformations for energy minimizations were obtained from the molecular dynamics simulations and from a systematic search of the conformational space available to the molecules. Several minimum energy backbone and side-chain conformations were found for each analogue. The effect of retro-inverso residues on hydrogen-bonding patterns and backbone conformations is discussed.     

Unusual (saw-like) temperature dependence of the rate of irreversible thermoinactivation of enzymes

An unusual ("zig-zag") temperature dependence of the rate of irreversible thermoinactivation of enzymes was observed for native and covalently modified alpha-chymotrypsin and trypsin. This dependence was characterized by alternation of plots with positive and negative apparent values of activation energy for the thermoinactivation process. A kinetic scheme which reflects the observed regularities in thermoinactivation for which the temperature-dependent conformational transition is an essential feature, is proposed. Fluorescence spectroscopy data suggest that the conformational transition predicted by the scheme is of the unfolding type. Substantial differences in thermostabilities of "high temperature" and "low temperature" conformations of enzymes may be due to different mechanisms of their irreversible thermoinactivation.     

Constructing ensembles of flexible fragments in native proteins by iterative stochastic elimination is relevant to protein-protein interfaces

We investigate the extent to which ensembles of flexible fragments (FF), generated by our loop conformational search method, include conformations that are near experimental and reflect conformational changes that these FFs undergo when binary protein-protein complexes are formed. Twenty-eight FFs, which are located in protein-protein interfaces and have different conformations in the bound structure (BS) and unbound structure (UbS) were extracted. The conformational space of these fragments in the BS and UbS was explored with our method which is based on the iterative stochastic elimination (ISE) algorithm. Conformational search of BSs generated bound ensembles and conformational search of UbSs produced unbound ensembles. ISE samples conformations near experimental (less than 1.05 A root mean square deviation, RMSD) for 51 out of the 56 examined fragments in the bound and unbound ensembles. In 14 out of the 28 unbound fragments, it also samples conformations within 1.05 A from the BS in the unbound ensemble. Sampling the bound conformation in the unbound ensemble demonstrates the potential biological relevance of the predicted ensemble. The 10 lowest energy conformations are the best choice for docking experiments, compared with any other 10 conformations of the ensembles. We conclude that generating conformational ensembles for FFs with ISE is relevant to FF conformations in the UbS and BS. Forming ensembles of the isolated proteins with our method prior to docking represents more comprehensively their inherent flexibility and is expected to improve docking experiments compared with results obtained by docking only UbSs.     

Direct assessment of CXCR4 mutant conformations reveals complex link between receptor structure and G(alpha)(i) activation

Ligand binding to G protein-coupled receptors (GPCRs) is thought to induce changes in receptor conformation that translate into activation of downstream effectors. The link between receptor conformation and activity is still insufficiently understood, as current models of GPCR activation fail to take an increasing amount of experimental data into account. To elucidate structure-function relationships in GPCR activation, we used bioluminescence resonance energy transfer to directly assess the conformation of mutants of the chemokine receptor CXCR4. We analyzed substitutions in the arginine cage DRY motif and in the conserved asparagine N(3.35)119, which are pivotal molecular switches for receptor conformation and activation. G(alpha)(i) activation of the mutants was either similar to wild-type CXCR4 (D133N, Y135A, and N119D) or resulted in loss of activity (R134A and N119K). Mutant N119S was constitutively active but further activated by agonist. Bioluminescence resonance energy transfer analysis suggested no simple correlation between conformational changes in response to ligand binding and activation of G(alpha)(i) by the mutants. Different conformations of active receptors were detected (for wild-type CXCR4, D133N, and N119S), suggesting that different receptor conformations are able to trigger G(alpha)(i) activity. Several conformations were also found for inactive mutants. These data provide biophysical evidence for different receptor conformations being active with respect to a single readout. They support models of GPCR structure-activity relationships that take this conformational flexibility of active receptors into account.     

Topography of the active site of the noradrenaline neuronal membrane carrier based on the theoretical conformation analysis of inhibitors of neuronal catecholamine uptake

All equilibrium conformations for nine ligands, essentially different in structure, of the noradrenaline carrier through the synaptosomal membrane of rat hypothalamus were calculated by semi-empirical method. Among these compounds were amphetamine, methylphenidate, tricyclic antidepressants. The conformational energy minimization was performed in the space of torsional and bond angles. Geometrical characteristics of the conformers were determined in the cartesian coordinate system fixed relative to the benzene ring and nitrogen atom of the ligand. The selection of biologically active (productive) conformations was made according to the following criteria: 1) low conformational energy; 2) similarity of the nitrogen atoms and phenyl rings spatial disposition in all ligands; 3) accessibility for intermolecular interactions of the same sides of functional groupings in all ligands. The above criteria enabled the productive conformations for all ligands to be chosen unambiguously. The productive conformation of noradrenaline was found to have the Ph-C-C-N fragment in perpendicular trans-conformation. A topographic model for the carrier active site was suggested, its components being the nucleophilic and two arylophilic groups situated against the most accessible sides of the functional moieties of the productively bound ligands.     

Main-chain conformational tendencies of amino acids

A Ramachandran plot is a visual representation of the main-chain conformational tendencies of an amino acid. Despite forty years of research, the shape of Ramachandran plots is still a matter of debate. The issue in making a Ramachandran plot based on experimental data is deciding whether sparse data represent genuine conformations. We present here a simple solution to settle the ambiguities of the sparse data, and explain how we verified the accuracies of our plots using an independent dataset. To obtain our results, we then measured the pair-wise distances of main-chain conformational tendencies among amino acids, and showed that the conformational relationships of amino acids are well preserved in a two-dimensional map, leading to the conclusion that the conformational diversity space of amino acids is largely two dimensional. We further noticed that amino acids in early and late evolutionary stages are located in different zones in the two-dimensional map. In addition to these conclusions, we here present an amino acid substitution table derived from experimental data.     

Monte Carlo calculations on the conformations of models for the glycopeptide linkage of glycoproteins

In order to search for probable conformations of the peptide, the amino acid side chain, and the carbohydrate linkage in glycoproteins, conformational energy surfaces of glycopeptide model compounds were studied by Monte Carlo methods using the Metropolis algorithm. The potential energies were composed of empirical energy functions which include nonbonded interactions, electrostatics, hydrogen bonding, and torsional energies specified by parameters which have been used for peptides. Calculations were performed on 1-N-acetyl-2-acetamido-beta-D-glucopyranosyl amine and the glycosylated dipeptide N-acetyl-delta-N-(2-acetamido-beta-D-glucopyranosyl)-L-asparaginyl-N'-methyl amide as models for N-glycosylated peptides and on methyl-2-acetamido-alpha-D-galactopyranoside as well as the glycosylated dipeptides N-acetyl-gamma-O-(2-acetamido-alpha-D-galactopyranosyl)-L-threonyl-N'-methyl amide and its seryl analog as models for O-glycosylated glycoproteins. The probable conformations of these compounds were analyzed by single-angle probability tables and by two-dimensional conformation density maps projected from the Markov chains which contained up to six independently varied conformational dihedral angles. The presence of high barriers to rotation required the use of search strategies which resulted in a rather low acceptance rate for new conformations in the Metropolis algorithm in order to avoid trapping of the Markov chain in local energy minima. This problem contributed to the failure of these calculations to attain complete convergence to the thermodynamic limit for the glycosylated dipeptide models in which six dihedral angles were independently varied. Analysis of the results shows that the conformational space available to the highly crowded axial glycosides of the alpha-O-GalNAc type is much more restricted than that for the N-asparaginyl glycopeptides. The most probable conformation for the O-glycosylated peptides is is a beta-turn while N-glycosylated peptides may be either in a beta-turn or an extended conformation.     

Refinement of the thrombin-bound structure of a hirudin peptide by a restrained electrostatically driven Monte Carlo method.

Energy refinement of the structure of a linear peptide, hirudin56-65, bound to thrombin was carried out using a conformational search method in combination with restrained minimization. Five conformations originated from nmr data and distance geometry calculations having a similar global folding pattern but quite different backbone conformations were used as the starting structures. As a result of this approach, a series of low-energy conformations compatible with a set of upper and lower bounds of interproton distances determined from transferred nuclear Overhauser effects were found. A comparison among the lowest energy conformations of each run showed that the combination of energy refinement plus distance constraints led to a very well-defined structure for both the backbone and the side chains of the last 7 residues of the polypeptide. Furthermore, the low-energy conformations generated with this technique contain a segment of 3(10)-helix involving the last 5 residues at the COOH terminal end.     

Kinetic and structural characterization of urease active site variants

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.