NO rebinding to myoglobin: a reactive molecular dynamics study

The rebinding of NO to myoglobin after photolysis is studied using the 'reactive molecular dynamics' method. In this approach the energy of the system is evaluated on two potential energy surfaces that include the heme-ligand interactions which change between liganded and unliganded myoglobin. This makes it possible to take into account in a simple way, the high dimensionality of the transition seam connecting the reactant and product states. The dynamics of the dissociated NO molecules are examined, and the geometrical and energetic properties of the transition seam are studied. Analysis of the frequency of recrossing shows that the height of the effective rebinding barrier is dependent on the time after photodissociation. This effect is due mainly to protein relaxation and may contribute to the experimentally observed non-exponential rebinding rate of NO, as has been suggested previously.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

Conformational analysis and molecular dynamics simulations of maltose

Constrained conformational energy minimizations have been used to calculate an adiabatic (Φ, ψ) potential energy surface for the disaccharide β-maltose. The inclusion of molecular flexibility in the conformational energy analysis of the disaccharide was found to significantly lower the barriers to conformational transitions, as has been observed previously for other systems. Several low energy wells were identified on the adiabatic surface which differ in energy by small amounts and with low absolute barriers separating them, indicating the possibility of a non-negligible equilibrium population distribution in each well. If such a distribution of conformations existed in the physical system, the conformation observed by NMR NOE measurements would thus be a “virtual” conformation. Molecular dynamics simulations of the motions of this molecule in vacuum were also conducted and indicate that the rate of relaxation of the molecule to the adiabatic surface may be slower than the typical timescale of conformational fluctuations. This effect is apparently due to an unphysical persistence of hydrogen bond patterns in vacuum which does not occur in aqueous solution. Trajectories undergoing transitions between wells were calculated and the effects of such conformational transitions upon the ensemble mean structure, such as might be observed in an NMR experiment, were demonstrated.     

Control of nitric oxide dynamics by guanylate cyclase in its activated state

Soluble guanylate cyclase (sGC) is the target of nitric oxide (NO) released by nitric-oxide synthase in endothelial cells, inducing an increase of cGMP synthesis in response. This heterodimeric protein possesses a regulatory subunit carrying a heme where NO binding occurs, while the second subunit harbors the catalytic site. The binding of NO and the subsequent breaking of the bond between the proximal histidine and the heme-Fe(2+) are assumed to induce conformational changes, which are the origin of the catalytic activation. At the molecular level, the activation and deactivation mechanisms are unknown, as is the dynamics of NO once in the heme pocket. Using ultrafast time-resolved absorption spectroscopy, we measured the kinetics of NO rebinding to sGC after photodissociation. The main spectral transient in the Soret band does not match the equilibrium difference spectrum of NO-liganded minus unliganded sGC, and the geminate rebinding was found to be monoexponential and ultrafast (tau = 7.5 ps), with a relative amplitude close to unity (0.97). These characteristics, so far not observed in other hemoproteins, indicate that NO encounters a high energy barrier for escaping from the heme pocket once the His-Fe(2+) bond has been cleaved; this bond does not reform before NO recombination. The deactivation of isolated sGC cannot occur by only simple diffusion of NO from the heme; therefore, several allosteric states may be inferred, including a desensitized one, to induce NO release. Thus, besides the structural change leading to activation, a consequence of the decoupling of the proximal histidine may also be to induce a change of the heme pocket distal geometry, which raises the energy barrier for NO escape, optimizing the efficiency of NO trapping. The non-single exponential character of the NO picosecond rebinding coexists only with the presence of the protein structure surrounding the heme, and the single exponential rate observed in sGC is very likely to be due to a closed conformation of the heme pocket. Our results emphasize the physiological importance of NO geminate recombination in hemoproteins like nitric-oxide synthase and sGC and show that the protein structure controls NO dynamics in a manner adapted to their function. This control of ligand dynamics provides a regulation at molecular level in the function of these enzymes.     

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.     

Role of heme iron coordination and protein structure in the dynamics and geminate rebinding of nitric oxide to the H93G myoglobin mutant: implications for nitric oxide sensors

The influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases ( approximately 10 and approximately 100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes.     

NO binding and dynamics in reduced heme-copper oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba(3) from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa(3), whereas strong rebinding on the 15-ns time scale was observed for CcO ba(3). The EPR characterizations show similar results for aa(3) at substoichiometric NO/enzyme ratios and for ba(3), indicating formation of a 6-coordinate heme-NO complex. The presence of a second NO molecule in the aa(3) active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a approximately 63 degrees rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB.     

Microseconds dynamics simulations of the outer-membrane protease T

Conformational fluctuations of enzymes may play an important role for substrate recognition and/or catalysis, as it has been suggested in the case of the protease enzymatic superfamily. Unfortunately, theoretically addressing this issue is a problem of formidable complexity, as the number of the involved degrees of freedom is enormous: indeed, the biological function of a protein depends, in principle, on all its atoms and on the surrounding water molecules. Here we investigated a membrane protease enzyme, the OmpT from Escherichia coli, by a hybrid molecular mechanics/coarse-grained approach, in which the active site is treated with the GROMOS force field, whereas the protein scaffold is described with a Go-model. The method has been previously tested against results obtained with all-atom simulations. Our results show that the large-scale motions and fluctuations of the electric field in the microsecond timescale may impact on the biological function and suggest that OmpT employs the same catalytic strategy as aspartic proteases. Such a conclusion cannot be drawn within the 10- to 100-ns timescale typical of current molecular dynamics simulations. In addition, our studies provide a structural explanation for the drop in the catalytic activity of two known mutants (S99A and H212A), suggesting that the coarse-grained approach is a fast and reliable tool for providing structure/function relationships for both wild-type OmpT and mutants.     

Elasticity-associated rebinding rate of molecular bonds between soft elastic media

A quantitative understanding of how cells interact with their extracellular matrix via molecular bonds is fundamental for many important processes in cell biology and engineering. In these interactions, the deformability of cells and matrix are usually comparable with that of the bonds, making their rebinding events globally coupled with the deformation states of whole systems. Unfortunately, this important principle is not realized or adopted in most conventional theoretical models for analyzing cellular adhesions. In this study, we considered a new theoretical model of a cluster of ligand-receptor bonds between two soft elastic bodies, in which the rebinding rates of ligands to receptors are described, by considering the deformation of the overall system under the influence of bond distributions. On the basis of theory of continuum and statistical mechanics, we obtained an elasticity-associated rebinding rate of open bonds in a closed analytical form that highly depends on the binding states and distributions of all other bonds as well as on the overall deformation energy stored in the elastic bodies and all closed bonds. On the basis of this elasticity-associated rebinding rate and by performing Monte Carlo simulations, we uncovered new mechanisms underlying the adhesion stability of molecular-bond clusters associated with deformable elastic bodies. Moreover, we revealed that the rebinding processes of molecular bonds is not only dependent on interfacial separation but is related to overall energy. This newly proposed rebinding rate may substantially improve our understanding of how cells adapt to their microenvironments by adjusting their mechanical properties through cytoskeleton remodeling.     

Molecular dynamics of native protein. I. Computer simulation of trajectories

The motion of the atoms in the small protein bovine pancreatic trypsin inhibitor has been simulated for about 60 picoseconds using two different potential energy functions. In one known as HHL the hydrogen bond is purely electrostatic, in the other, known as L79, the hydrogen bond is a directional O . . . H interaction. The energy parameters and techniques used to obtain an accurate, well-equilibrated trajectory are described in detail. The trajectories calculated here with either potential are superior to those obtained in previous simulations on the same protein in that they treat hydrogen bonding realistically and remain closer to the native X-ray structure. Comparison of the two trajectories shows that the potential energy parameters have a significant effect on the shift from the X-ray structure, the distribution of (phi, psi) torsion angles, the pattern of hydrogen bonds and the accessible surface area of individual residues. The L79 potential with directional hydrogen bonds is used to simulate a longer 132 picosecond trajectory that is analysed in the accompanying paper.     

Geminate recombination of nitric oxide to endothelial nitric-oxide synthase and mechanistic implications.

The nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and NO through consumption of oxygen bound to the heme. Because NO is produced close to the heme and may bind to it, its subsequent role in a regulatory mechanism should be scrutinized. We therefore examined the kinetics of NO rebinding after photodissociation in the heme pocket of human endothelial NOS by means of time-resolved absorption spectroscopy. We show that geminate recombination of NO indeed occurs and that this process is strongly modulated by L-Arg. This NO rebinding occurs in a multiphasic fashion and spans over 3 orders of magnitude. In both ferric and ferrous states of the heme, a fast nonexponential picosecond geminate rebinding first takes place followed by a slower nanosecond phase. The rates of both phases decreased, whereas their relative amplitudes are changed by the presence of L-Arg; the overall effect is a slow down of NO rebinding. For the isolated oxygenase domain, the picosecond rate is unchanged, but the relative amplitude of the nanosecond binding decreased. We assigned the nanosecond kinetic component to the rebinding of NO that is still located in the protein core but not in the heme pocket. The implications for a mechanism of regulation involving NO binding are discussed.     

Relationship between protein structural fluctuations and rebinding dynamics in ferric haem nitrosyls

The interaction of nitric oxide (NO) with haem proteins is widespread in biology. In the current paper, we present the first ultrafast 2D-IR (two-dimensional infrared) spectroscopic analysis of haem nitrosylation, which has been combined with time-resolved IR pump-probe studies to investigate the relationship between equilibrium vibrational dynamics of the haem environment and ligand rebinding behaviour following photolysis of NO from the Fe(III)-NO site. Studies of two haem proteins, Mb (myoglobin) and Cc (cytochrome c), which play different physiological roles, reveal marked contrasts in the ultrafast fluctuations of the protein pockets containing the haem, showing that the Mb pocket is somewhat more flexible than that of Cc. This correlates strongly with slower observed photolysis rebinding kinetics of Mb-NO compared with Cc-NO, and indicates a direct link between ultrafast fluctuations and biological functionality. Furthermore, this indicates the validity of linear response theories in relation to protein ligand binding. Finally, 2D-IR shows that Cc-NO displays two distinct structural sub-sites at room temperature that do not exchange on the timescales accessible via the NO vibrational lifetime.     

Quaternary structure controls ligand dynamics in soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β(1)(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β(1)(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β(1)(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates k(on) = 0.075 ± 0.01 × 10(6) M(-1) · S(-1) for sGC and 0.83 ± 0.1 × 10(6) M(-1) · S(-1) for β(1)(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β(1)(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe(2+)-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit.     

Energetics and dynamics of constrained actin filament bundling

The formation of filopodia-like bundles from a dendritic actin network has been observed to occur in vitro as a result of branching induced by Arp2/3 complex. We study both the energetics and dynamics of actin filament bundling in such a network to evaluate their relative importance in bundle formation processes. Our model considers two semiflexible actin filaments fixed at one end and free at the other, described using a normal-mode approximation. This model is studied by both Brownian dynamics and free-energy minimization methods. Remarkably, even short filaments can bundle at separations comparable to their lengths. In the dynamic simulations, we evaluate the time required for the filaments to interact and bind, and examine the dependence of this bundling time on the filament length, the distance between the filament bases, and the cross-linking energy. In most cases, bundling occurs in a second or less. Beyond a certain critical distance, we find that the bundling time increases very rapidly with increasing interfilament separation and/or decreasing filament length. For most of the cases we have studied, the energetics results for this critical distance are similar to those obtained from dynamics simulations run for 10 s, suggesting that beyond this timescale, energetics, rather than kinetic constraints, determine whether or not bundling occurs. Over a broad range of conditions, we find that the times required for bundling from a network are compatible with experimental observations.     

Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water

The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using “realistic” potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.     

Ligand dynamics and early signaling events in the heme domain of the sensor protein Dos from Escherichia coli

In the heme-based sensor Dos from Escherichia coli, the ferrous heme is coordinated by His-77 and Met-95. The latter residue is replaced upon oxygen binding or oxidation of the heme. Here we investigate the early signaling processes upon dissociation of the distal ligand using ultrafast spectroscopy and site-directed mutagenesis. Geminate CO rebinding to the heme domain DosH appears insensitive to replacement of Met-95, in agreement with the notion that this residue is oriented out of the heme pocket in the presence of external ligands. A uniquely slow 35-ps phase in rebinding of the flexible methionine side chain after dissociation from ferrous DosH is completely abolished in rebinding of the more rigid histidine side chain in the M95H mutant protein, where only the 7-ps phase, common to all 6-coordinate heme proteins, is observed. Temperature-dependence studies indicate that all rebinding of internal and external ligands is essentially barrierless, but that CfigsO escape from the heme pocket is an activated process. Solvent viscosity studies combined with molecular dynamics simulations show that there are two configurations in the ferrous 6-coordinate protein, involving two isomers of the Met-95 side chain, of which the structural changes extend to the solvent-exposed backbone, which is part of the flexible FG loop. One of these configurations has considerable motional freedom in the Met-95-dissociated state. We suggest that this configuration corresponds to an early signaling intermediate state, is responsible for the slow rebinding, and allows small ligands in the protein to efficiently compete for binding with the heme.     

Accelerated molecular dynamics simulations of the octopamine receptor using GPUs: discovery of an alternate agonist‐binding position

Octopamine receptors (OARs) perform key biological functions in invertebrates, making this class of G‐protein coupled receptors (GPCRs) worth considering for insecticide development. However, no crystal structures and very little research exists for OARs. Furthermore, GPCRs are large proteins, are suspended in a lipid bilayer, and are activated on the millisecond timescale, all of which make conventional molecular dynamics (MD) simulations infeasible, even if run on large supercomputers. However, accelerated Molecular Dynamics (aMD) simulations can reduce this timescale to even hundreds of nanoseconds, while running the simulations on graphics processing units (GPUs) would enable even small clusters of GPUs to have processing power equivalent to hundreds of CPUs. Our results show that aMD simulations run on GPUs can successfully obtain the active and inactive state conformations of a GPCR on this reduced timescale. Furthermore, we discovered a potential alternate active‐state agonist‐binding position in the octopamine receptor which has yet to be observed and may be a novel GPCR agonist‐binding position. These results demonstrate that a complex biological system with an activation process on the millisecond timescale can be successfully simulated on the nanosecond timescale using a simple computing system consisting of a small number of GPUs. Proteins 2016; 84:1480–1489. © 2016 Wiley Periodicals, Inc.     

Energy landscape distortions and the mechanical unfolding of proteins

Molecular simulations and an energy landscape analysis are used to examine the stretching of a model protein. A mapping of the energy landscape shows that stretching the protein causes energy minima and energy barriers to flatten out and disappear, and new energy minima to be created. The implications of these landscape distortions depend on the timescale regime under which the protein is stretched. When the timescale for thermally activated processes is longer than the timescale of stretching, the disappearances of energy barriers provide the mechanism for protein unfolding. When the timescale for thermally activated processes is shorter than the timescale of stretching, the landscape distortions influence the stretching process by changing the number and types of energy minima in which the system can exist.     

Time-averaged order parameter restraints in molecular dynamics simulations

A method is described that allows experimental \(S^2\) order parameters to be enforced as a time-averaged quantity in molecular dynamics simulations. The two parameters that characterize time-averaged restraining, the memory relaxation time and the weight of the restraining potential energy term in the potential energy function used in the simulation, are systematically investigated based on two model systems, a vector with one end restrained in space and a pentapeptide. For the latter it is shown that the backbone N–H order parameter of individual residues can be enforced such that the spatial fluctuations of quantities depending on atomic coordinates are not significantly perturbed. The applicability to realistic systems is illustrated for the B3 domain of protein G in aqueous solution.