Molecular dynamics simulations of lignin peroxidase in solution

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.     

Locally accessible conformations of proteins: multiple molecular dynamics simulations of crambin.

Multiple molecular dynamics (MD) simulations of crambin with different initial atomic velocities are used to sample conformations in the vicinity of the native structure. Individual trajectories of length up to 5 ns sample only a fraction of the conformational distribution generated by ten independent 120 ps trajectories at 300 K. The backbone atom conformational space distribution is analyzed using principal components analysis (PCA). Four different major conformational regions are found. In general, a trajectory samples only one region and few transitions between the regions are observed. Consequently, the averages of structural and dynamic properties over the ten trajectories differ significantly from those obtained from individual trajectories. The nature of the conformational sampling has important consequences for the utilization of MD simulations for a wide range of problems, such as comparisons with X-ray or NMR data. The overall average structure is significantly closer to the X-ray structure than any of the individual trajectory average structures. The high frequency (less than 10 ps) atomic fluctuations from the ten trajectories tend to be similar, but the lower frequency (100 ps) motions are different. To improve conformational sampling in molecular dynamics simulations of proteins, as in nucleic acids, multiple trajectories with different initial conditions should be used rather than a single long trajectory.     

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.     

Essential dynamics of DNA containing a cis.syn cyclobutane thymine dimer lesion.

Conformational properties of a UV-damaged DNA decamer containing a cis.syn cyclobutane thymine dimer (PD) have been investigated by molecular dynamics (MD) simulations. Results from MD simulations of the damaged decamer DNA show a kink of approximately 21.7 degrees at the PD damaged site and a disruption of H bonding between the 5'-thymine of the PD and its complementary adenine. However, no extra-helical flipping of the 3'-adenine complementary to the PD was observed. Comparison to two undamaged DNA decamers, one with the same sequence and the other with an AT replacing the TT sequence, indicates that these properties are specific to the damaged DNA. Essential dynamics (ED) derived from the MD trajectories of the three DNAs show that the backbone phosphate between the two adenines complementary to the PD of the damaged DNA has considerably larger mobility than the rest of the molecule and occurs only in the damaged DNA. As observed in the crystal structure of T4 endonuclease V in a complex with the damaged DNA, the interaction of the enzyme with the damaged DNA can lead to bending along the flexible joint and to induction of adenine flipping into an extra-helical position. Such motions may play an important role in damage recognition by repair enzymes.     

Molecular dynamics and IR spectroscopy in investigation of phase transitions in molecular crystal 4,4'-bis(11-hydroxy-1-undecyloxy)biphenyl

Molecular dynamics (MD) simulations combined with temperature-dependent IR spectroscopic measurements were used to study phase transitions in molecular crystals of the mesogenic diol 4,4'-bis(11-hydroxy-1-undecyloxy)biphenyl. DSC measurements revealed four phase transitions in this molecular crystal at approximately 327.1 K, 389.8 K, 419.1 K and 431.9 K. Analysis of the dynamic trajectories at temperatures of 300 K, 360 K, 400 K and 480 K revealed changes in conformation of the mesogenic diol molecules and consequently changes in crystal packing and crystal structure in the temperature range 300-480 K and enabled us to understand the mechanism of the phase transitions.     

Ensemble MD simulations restrained via crystallographic data: Accurate structure leads to accurate dynamics

Currently, the best existing molecular dynamics (MD) force fields cannot accurately reproduce the global free‐energy minimum which realizes the experimental protein structure. As a result, long MD trajectories tend to drift away from the starting coordinates (e.g., crystallographic structures). To address this problem, we have devised a new simulation strategy aimed at protein crystals. An MD simulation of protein crystal is essentially an ensemble simulation involving multiple protein molecules in a crystal unit cell (or a block of unit cells). To ensure that average protein coordinates remain correct during the simulation, we introduced crystallography‐based restraints into the MD protocol. Because these restraints are aimed at the ensemble‐average structure, they have only minimal impact on conformational dynamics of the individual protein molecules. So long as the average structure remains reasonable, the proteins move in a native‐like fashion as dictated by the original force field. To validate this approach, we have used the data from solid‐state NMR spectroscopy, which is the orthogonal experimental technique uniquely sensitive to protein local dynamics. The new method has been tested on the well‐established model protein, ubiquitin. The ensemble‐restrained MD simulations produced lower crystallographic R factors than conventional simulations; they also led to more accurate predictions for crystallographic temperature factors, solid‐state chemical shifts, and backbone order parameters. The predictions for ¹⁵N relaxation rates are at least as accurate as those obtained from conventional simulations. Taken together, these results suggest that the presented trajectories may be among the most realistic protein MD simulations ever reported. In this context, the ensemble restraints based on high‐resolution crystallographic data can be viewed as protein‐specific empirical corrections to the standard force fields.     

Going beyond Clustering in MD Trajectory Analysis: An Application to Villin Headpiece Folding

Recent advances in computing technology have enabled microsecond long all-atom molecular dynamics (MD) simulations of biological systems. Methods that can distill the salient features of such large trajectories are now urgently needed. Conventional clustering methods used to analyze MD trajectories suffer from various setbacks, namely (i) they are not data driven, (ii) they are unstable to noise and changes in cut-off parameters such as cluster radius and cluster number, and (iii) they do not reduce the dimensionality of the trajectories, and hence are unsuitable for finding collective coordinates. We advocate the application of principal component analysis (PCA) and a non-metric multidimensional scaling (nMDS) method to reduce MD trajectories and overcome the drawbacks of clustering. To illustrate the superiority of nMDS over other methods in reducing data and reproducing salient features, we analyze three complete villin headpiece folding trajectories. Our analysis suggests that the folding process of the villin headpiece is structurally heterogeneous.     

Simulating proteins at constant pH: An approach combining molecular dynamics and Monte Carlo simulation

For the structure and function of proteins, the pH of the solution is one of the determining parameters. Current molecular dynamics (MD) simulations account for the solution pH only in a limited way by keeping each titratable site in a chosen protonation state. We present an algorithm that generates trajectories at a Boltzmann distributed ensemble of protonation states by a combination of MD and Monte Carlo (MC) simulation. The algorithm is useful for pH-dependent structural studies and to investigate in detail the titration behavior of proteins. The method is tested on the acidic residues of the protein hen egg white lysozyme. It is shown that small structural changes may have a big effect on the pK(A) values of titratable residues.     

Collective dynamics of EcoRI-DNA complex by elastic network model and molecular dynamics simulations

Anisotropic network model (ANM) is used to analyze the collective motions of restriction enzyme EcoRI in free form and in complex with DNA. For comparison, three independent molecular dynamics (MD) simulations, each of 1.5 ns duration, are also performed for the EcoRI-DNA complex in explicit water. Although high mobility (equilibrium fluctuations) of inner and outer loops that surround the DNA is consistent in both methods and experiments, MD runs sample different conformational subspaces from which reliable collective dynamics cannot be extracted. However, ANM employed on different conformations from MD simulations indicates very similar collective motions. The stems of the inner loops are quite immobile even in the free enzyme and form a large, almost fixed, pocket for DNA binding. As a result, the residues that make specific and non-specific interactions with the DNA exhibit very low fluctuations in the free enzyme. The vibrational entropy difference between the EcoRI complex and free protein + unkinked DNA is positive (favorable), which may partially counteract the unfavorable enthalpy difference of DNA kink formation. Dynamic domains in EcoRI complex and cross-correlations between residue fluctuations indicate possible means of communication between the distal active sites.     

Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation

The ubiquitin associated domain of p62 is a small three-helix bundle of approximately 50 residues that mediates the recognition of polyubiquitin chains and ubiquitylated substrates. The solution structure of a 52 residue construct containing this domain has been characterized using heteronuclear nuclear magnetic resonance (NMR) methods. The resulting ensemble of NMR-derived structures was used in molecular dynamics (MD) simulations to investigate the equilibrium conformation and dynamics of this domain. NOE and (15)N relaxation data have been used to validate the structural ensemble produced by the MD simulations and show a good correlation for residues in regions of secondary structure. A similar approach was taken using an ensemble of structures from the MD simulations to calculate electronic circular dichroism (CD) and IR spectra from first principles with an encouraging correlation with the experimental CD and IR data.     

Fuzzy cluster analysis of molecular dynamics trajectories.

We propose fuzzy clustering as a method to analyze molecular dynamics (MD) trajectories, especially of proteins and polypeptides. A fuzzy cluster analysis locates classes of similar three-dimensional conformations explored during a molecular dynamics simulation. The method can be readily applied to results from both equilibrium and nonequilibrium simulations, with clustering on either global or local structural parameters. The potential of this technique is illustrated by results from fuzzy cluster analyses of trajectories from MD simulations of various fragments of human parathyroid hormone (PTH). For large molecules, it is more efficient to analyze the clustering of root-mean-square distances between conformations comprising the trajectory. We found that the results of the clustering analysis were unambiguous, in terms of the optimal number of clusters of conformations, for the majority of the trajectories examined. The conformation closest to the cluster center can be chosen as being representative of the class of structures making up the cluster, and can be further analyzed, for example, in terms of its secondary structure. The CPU time used by the cluster analysis was negligible compared to the MD simulation time.     

Weight average approaches for predicting dynamical properties of biomolecules

Recent advances in atomistic molecular dynamics (MD) simulations of biomolecules allow us to explore their conformational spaces widely, observing large-scale conformational fluctuations or transitions between distinct structures. To reproduce or refine experimental data using MD simulations, structure ensembles, which are characterized by multiple structures and their statistical weights on the rugged free-energy landscapes, are often used. Here, we summarize weight average approaches for various experimental measurements. Weight average approaches are now applied to hybrid quantum mechanics/molecular mechanics MD simulations to predict fast vibrational motions in a protein with a high accuracy for better understanding of molecular functions from atomic structures.     

A short guide for molecular dynamics simulations of RNA systems

As a result of important methodological advances and of the rapid growth of experimental data, the number of molecular dynamics (MD) simulations related to RNA systems has significantly increased. However, such MD simulations are not straightforward and great care has to be exerted during the setup stage in order to choose the appropriate MD package, force fields and ionic conditions. Furthermore, the choice and a correct evaluation of the main characteristics of the starting structure are primordial for the generation of informative and reliable MD trajectories since experimental structures are not void of inaccuracies and errors. The aim of this review is to provide, through numerous examples, practical guidelines for the setup of MD simulations, the choice of ionic conditions and the detection and correction of experimental inaccuracies in order to start MD simulations of nucleic acid systems under the best auspices.     

Protein conformational dynamics of crambin in crystal, solution and in the trajectories of molecular dynamics simulations

Atomic displacement parameters — B factors of the eight crambin crystal structures obtained at 0.54–1.5 Å resolution and temperatures of 100–293 K have been analyzed. The comparable contributions to the B factor values are the intramolecular motions which are modeled by the harmonic vibration calculations and derived from the molecular dynamics simulation (MD) as well as rigid body changes in the position of a protein molecule as a whole. In solution for the average NMR structure of crambin the amplitudes of the backbone atomic fluctuations of the most residues of the segments with the regular backbone conformations are close to the amplitudes of the small scale harmonic vibrations. For the same residues the probability of the medium scale fluctuations fixed by the hydrogen exchange method is very low. The restricted conformational mobility of those segments is coupled with the depressed amplitudes of the fluctuation changes of the tertiary structure registered by the residue accessibility changes in an ensemble of NMR structures that forms the average NMR structure of crambin. The amplitudes of temperature fluctuations of backbone atoms and the tertiary structure raise in the segment with the irregular conformations, turn and loops. In the same segments the amplitudes of the calculated harmonic vibrations also increase, but to a lesser extent and especially in the interhelical loop with the most strong and complicated fluctuation changes of the backbone conformation. In solution for the NMR structure in this loop the conformational transitions occur between the conformational substates separated by the energy barriers, but they are not observed even in the long 100 ns trajectories from the MD simulation of crambin. These strong local fluctuation changes of the structure may play a key role in the protein functioning and modern performance improvements in the MD simulation techniques are oriented to increase the probability of protein appearance in the trajectories from the MD simulations.     

Atomistic simulations reveal bubbles, kinks and wrinkles in supercoiled DNA

Although DNA is frequently bent and supercoiled in the cell, much of the available information on DNA structure at the atomistic level is restricted to short linear sequences. We report atomistic molecular dynamics (MD) simulations of a series of DNA minicircles containing between 65 and 110 bp which we compare with a recent biochemical study of structural distortions in these tight DNA loops. We have observed a wealth of non-canonical DNA structures such as kinks, denaturation bubbles and wrinkled conformations that form in response to bending and torsional stress. The simulations show that bending alone is sufficient to induce the formation of kinks in circles containing only 65 bp, but we did not observe any defects in simulations of larger torsionally relaxed circles containing 110 bp over the same MD timescales. We also observed that under-winding in minicircles ranging in size from 65 to 110 bp leads to the formation of single stranded bubbles and wrinkles. These calculations are used to assess the ability of atomistic MD simulations to determine the structure of bent and supercoiled DNA.     

Molecular dynamic simulations of environment and sequence dependent DNA conformations: the development of the BMS nucleic acid force field and comparison with experimental results

Molecular dynamic (MD) simulations using the BMS nucleic acid force field produce environment and sequence dependent DNA conformations that closely mimic experimentally derived structures. The parameters were initially developed to reproduce the potential energy surface, as defined by quantum mechanics, for a set of small molecules that can be used as the building blocks for nucleic acid macromolecules (dimethyl phosphate, cyclopentane, tetrahydrofuran, etc.). Then the dihedral parameters were fine tuned using a series of condensed phase MD simulations of DNA and RNA (in zero added salt, 4M NaCl, and 75% ethanol solutions). In the tuning process the free energy surface for each dihedral was derived from the MD ensemble and fitted to the conformational distributions and populations observed in 87 A- and B-DNA x-ray and 17 B-DNA NMR structures. Over 41 nanoseconds of MD simulations are presented which demonstrate that the force field is capable of producing stable trajectories, in the correct environments, of A-DNA, double stranded A-form RNA, B-DNA, Z-DNA, and a netropsin-DNA complex that closely reproduce the experimentally determined and/or canonical DNA conformations. Frequently the MD averaged structure is closer to the experimentally determined structure than to the canonical DNA conformation. MD simulations of A- to B- and B- to A-DNA transitions are also shown. A-DNA simulations in a low salt environment cleanly convert into the B-DNA conformation and converge into the RMS space sampled by a low salt simulation of the same sequence starting from B-DNA. In MD simulations using the BMS force field the B-form of d(GGGCCC)2 in a 75% ethanol solution converts into the A-form. Using the same methodology, parameters, and conditions the A-form of d(AAATTT)2 correctly converts into the B-DNA conformation. These studies demonstrate that the force field is capable of reproducing both environment and sequence dependent DNA structures. The 41 nanoseconds (nsec) of MD simulations presented in this paper paint a global picture which suggests that the DNA structures observed in low salt solutions are largely due to the favorable internal energy brought about by the nearly uniform screening of the DNA electrostatics. While the conformations sampled in high salt or mixed solvent environments occur from selective and asymmetric screening of the phosphate groups and DNA grooves, respectively, brought about by sequence induced ion and solvent packing.     

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.     

Principal component and normal mode analysis of proteins; a quantitative comparison using the GroEL subunit

Principal component analysis (PCA) and normal mode analysis (NMA) have emerged as two invaluable tools for studying conformational changes in proteins. To compare these approaches for studying protein dynamics, we have used a subunit of the GroEL chaperone, whose dynamics is well characterized. We first show that both PCA on trajectories from molecular dynamics (MD) simulations and NMA reveal a general dynamical behavior in agreement with what has previously been described for GroEL. We thus compare the reproducibility of PCA on independent MD runs and subsequently investigate the influence of the length of the MD simulations. We show that there is a relatively poor one-to-one correspondence between eigenvectors obtained from two independent runs and conclude that caution should be taken when analyzing principal components individually. We also observe that increasing the simulation length does not improve the agreement with the experimental structural difference. In fact, relatively short MD simulations are sufficient for this purpose. We observe a rapid convergence of the eigenvectors (after ca. 6 ns). Although there is not always a clear one-to-one correspondence, there is a qualitatively good agreement between the movements described by the first five modes obtained with the three different approaches; PCA, all-atoms NMA, and coarse-grained NMA. It is particularly interesting to relate this to the computational cost of the three methods. The results we obtain on the GroEL subunit contribute to the generalization of robust and reproducible strategies for the study of protein dynamics, using either NMA or PCA of trajectories from MD simulations.     

Disruption and formation of surface salt bridges are coupled to DNA binding by the integration host factor: a computational analysis

Revealing the thermodynamic driving force of protein-DNA interactions is crucial to the understanding of factors that dictate the properties and function of protein-DNA complexes. For the binding of DNA to DNA-wrapping proteins, such as the integration host factor (IHF), Record and co-workers proposed that the disruption of a large number of preexisting salt bridges is coupled with the binding process [Holbrook, J. A., et al. (2001) J. Mol. Biol. 310, 379]. To test this proposal, we have conducted explicit solvent MD simulations (multiple ～25-50 ns trajectories for each salt concentration) to examine the behavior of charged residues in IHF, especially concerning their ability to form salt bridges at different salt concentrations. Of the 17 cationic residues noted by Record and co-workers, most are engaged in salt bridge interactions for a significant portion of the trajectories, especially in the absence of salt. This observation suggests that, from a structural point of view, their proposal is plausible. However, the complex behaviors of charged residues observed in the MD simulations also suggest that the unusual thermodynamic characteristics of IHF-DNA binding likely arise from the interplay between complex dynamics of charged residues both in and beyond the DNA binding site. Moreover, a comparison of MD simulations at different salt concentrations suggests that the strong dependence of the IHF-DNA binding enthalpy on salt concentration may not be due to a significant decrease in the number of stable salt bridges in apo IHF at high salt concentrations. In addition to the Hofmeister effects quantified in more recent studies of IHF-DNA binding, we recommend consideration of the variation of the enthalpy change of salt bridge disruption at different salt concentrations. Finally, the simulation study presented here explicitly highlights the fact that the electrostatic properties of DNA-binding proteins can be rather different in the apo and DNA-bound states, which has important implications for the design of robust methods for predicting DNA binding sites in proteins.     

Classical molecular interaction potentials: improved setup procedure in molecular dynamics simulations of proteins.

The latest version of the classical molecular interaction potential (CMIP) has the ability to predict the position of crystallographic waters in several proteins with great accuracy. This article analyzes the ability of the CMIP functional to improve the setup procedure of the molecular system in molecular dynamics (MD) simulations of proteins. To this end, the CMIP strategy is used to include both water molecules and counterions in different protein systems. The structural details of the configurations sampled from trajectories obtained using the CMIP setup procedure are compared with those obtained from trajectories derived from a standard equilibration process. The results show that standard MD simulations can lead to artifactual results, which are avoided using the CMIP setup procedure. Because the CMIP is easy to implement at a low computational cost, it can be very useful in obtaining reliable MD trajectories.