The Role of Gln61 in HRas GTP hydrolysis: a quantum mechanics/molecular mechanics study

Activation of the water molecule involved in GTP hydrolysis within the HRas·RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln(61) will stabilize the transient OH(-) and H(3)O(+) molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant.     

The implementation of slab geometry for membrane-channel molecular dynamics simulations

Slab geometric boundary conditions are applied in the molecular dynamics simulation of a simple membrane-channel system. The results of the simulation were compared to those of an analogous system using normal three-dimensional periodic boundary conditions. Analysis of the dynamics and electrostatics of the system show that slab geometric periodicity eliminates the artificial bulk water orientational polarization that is present while using normal three-dimensional periodicity. Furthermore, even though the water occupancy and volume of our simple channel is the same when using either method, the electrostatic properties are considerably different when using slab geometry. In particular, the orientational polarization of water is seen to be different in the interior of the channel. This gives rise to a markedly different electric field within the channel. We discuss the implications of slab geometry for the future simulation of this type of system and for the study of channel transport properties.     

Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly

A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.     

A quantum mechanics/molecular mechanics study of the tyrosine residue, TyrD, of Photosystem II

QM/MM calculations have been used to monitor the oxidation of the D2-Tyr160, Tyr_D, residue involved in redox reactions in Photosystem II. The results indicate that in the reduced form the residue is involved in hydrogen bond donation via its phenolic head group to the τ-nitrogen of the neighboring D2-His189 residue. Oxidation to form the radical is accompanied by spontaneous transfer of the phenolic hydrogen to the τ-nitrogen of D2-His189 leading to the formation of a tyrosyl-imidazolium ion complex. Deprotonation of the imidazolium ion leads to the formation of a tyrosyl-imidazole neutral hydrogen-bonded complex. Comparison of calculated and experimental hyperfine coupling tensors and g-tensors suggests that the neutral imidazole complex is formed at physiological temperatures while the imidazolium complex may be stabilized at cryogenic temperatures.     

Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.     

Determinants of reactivity and selectivity in soluble epoxide hydrolase from quantum mechanics/molecular mechanics modeling

Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds.     

The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes

In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium.     

Assembly of lipoprotein particles revealed by coarse-grained molecular dynamics simulations

High-density lipoproteins (HDL) function as cholesterol transporters, facilitating the removal of excess cholesterol from the body. Due to the heterogeneity of native HDL particles (both in size and shape), the details on how these protein-lipid particles form and the structure they assume in their lipid-associated states are not well characterized. We report here a study of the self-assembly of discoidal HDL particles using coarse-grained (CG) molecular dynamics. The microsecond simulations reveal the self-assembly of HDL particles from disordered protein-lipid complexes to form structures containing many of the features of the generally accepted double-belt model for discoidal HDL particles. HDL assembly is found to proceed in two broad steps, aggregation of proteins and lipids driven by the hydrophobic effect which occurs on a approximately 1 micros time scale, followed by the optimization of the protein structure driven by increasingly specific protein-protein interactions.     

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.     

A quantum mechanics/molecular mechanics study of the catalytic mechanism of the thymidylate synthase

A theoretical study of the molecular mechanism of the thymidylate synthase-catalyzed reaction has been carried out using hybrid quantum mechanics/molecular mechanics methods. We have examined all of the stationary points (reactants, intermediates, transition structures, and products) on the multidimensional potential energy surfaces for the multistep enzymatic process. The characterization of these relevant structures facilitates the gaining of insight into the role of the different residues in the active site. Furthermore, analysis of the full energy profile has revealed that the step corresponding to the reduction of the exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), forming dTMP and 7,8-dihydrofolate (H2folate), is the rate-limiting step, in accordance with the experimental data. In this step, the hydride transfer and the scission of an overall conserved active site cysteine residue (Cys146 in Escherichia coli) take place in a concerted but very asynchronous way. These findings have also been tested with primary and secondary deuterium, tritium, and sulfur kinetic isotope effects, and the calculations have been compared to experimental data. Finally, the incorporation of high-level quantum mechanical corrections to the semiempirical AM1 Hamiltonian into our hybrid scheme has allowed us to obtain reasonable values of the energy barrier for the rate-limiting step. The resulting picture of the complete multistep enzyme mechanism that is obtained reveals several new features of substantial mechanistic interest.     

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Catalytic mechanism of aldose reductase studied by the combined potentials of quantum mechanics and molecular mechanics

The catalytic reduction of

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-glyceraldehyde to glycerol by aldose reductase has been investigated with the combined potentials of quantum mechanics (QM) and molecular mechanics (MM) to resolve the question of whether Tyr48 or His110 serves as the proton donor during catalysis. Site directed mutagenesis studies favor Tyr48 as the proton donor while the presence of a water channel linking the Nδ1 of His110 to the bulk solvent suggests that His110 is the proton donor. Utilizing the combined potentials of QM and MM, the binding mode of substrate

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-glyceraldehyde was investigated by optimizing the local geometry of Asp43, Lys77, Tyr48, His110 and NADPH at the active site of aldose reductase. Reaction pathways for the reduction of

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-glyceraldehyde to glycerol were then constructed by treating both Tyr48 and His110 as proton donors. Comparison of energetics obtained from the reaction pathways suggests His110 to be the proton donor. Based on these findings, a reduction mechanism of

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-glyceraldehyde to glycerol is described.     

Hybrid quantum mechanical/molecular mechanical molecular dynamics simulations of HIV-1 integrase/inhibitor complexes

Human immunodeficiency virus (HIV)-1 integrase (IN) is an attractive target for development of acquired immunodeficiency syndrome chemotherapy. In this study, conventional and coupled quantum mechanical and molecular mechanical (QM/MM) molecular dynamics (MD) simulations of HIV-1 IN complexed with 5CITEP (IN-5CITEP) were carried out. In addition to differences in the bound position of 5CITEP, significant differences at the two levels of theory were observed in the metal coordination geometry and the areas involving residues 116-119 and 140-166. In the conventional MD simulation, the coordination of Mg(2+) was found to be a near-perfect octahedral geometry whereas a distorted octahedral complex was observed in QM/MM. All of the above reasons lead to a different pattern of protein-ligand salt link formation that was not observed in the classical MD simulation. Furthermore to provide a theoretical understanding of inhibition mechanisms of 5CITEP and its derivative (DKA), hybrid QM/MM MD simulations of the two complexes (IN-5CITEP and IN-DKA) have been performed. The results reveal that areas involving residues 60-68, 116-119, and 140-149 were substantially different among the two systems. The two systems show similar pattern of metal coordination geometry, i.e., a distorted octahedron. In IN-DKA, both OD1 and OD2 of Asp-64 coordinate the Mg(2+) in a monodentate fashion whereas only OD1 is chelated to the metal as observed in IN-5CITEP. The high potency of DKA as compared to 5CITEP is supported by a strong salt link formed between its carboxylate moiety and the ammonium group of Lys-159. Detailed comparisons between HIV-1 IN complexed with DKA and with 5CITEP provide information about ligand structure effects on protein-ligand interactions in particular with the Lys-159. This is useful for the design of new selective HIV-1 IN inhibitors.     

Structure of a transient intermediate for GTP hydrolysis by ras

The flexibility of the conserved 57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the 57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.     

The nature of tryptophan radicals involved in the long-range electron transfer of lignin peroxidase and lignin peroxidase-like systems: Insights from quantum mechanical/molecular mechanics simulations

A catalytically active tryptophan radical has been demonstrated to be involved in the long-range electron transfer to the heme cofactor of lignin peroxidase (LiP) from Phanerochaete chrysosporium although no direct detection by EPR spectroscopy of the tryptophan radical intermediate has been reported to date. An engineering-based approach has been used to manipulate the microenvironment of the redox-active tryptophan site in LiP and Coprinus cinereus Peroxidase (CiP), allowing the direct evidence of the tryptophan radical species. In light of the newly available EPR experimental data, we performed a quantum mechanical/molecular mechanics computational study to characterize the tryptophan radicals in the above protein matrices as well as in pristine LiP. The nature of the tryptophan radicals is discussed together with the analysis of their environment with the aim of understanding the different behavior of pristine LiP in comparison with that of LiP and CiP variants.