Molecular dynamics simulations of papilloma virus E2 DNA sequences: dynamical models for oligonucleotide structures in solution

The specificity of papilloma virus E2 protein-DNA binding depends critically upon the sequence of a region of the DNA not in direct contact with the protein, and represents one of the simplest known examples of indirect readout. A detailed characterization of this system in solution is important to the further investigation hypothesis of a structural code for DNA recognition by regulatory proteins. In the crystalline state, the E2 DNA oligonucleotide sequence, d(ACCGAATTCGGT), exhibits three different structural forms. We report herein studies of the structure of E2 DNA in solution based on a series of molecular dynamics (MD) simulations including counterions and water, utilizing both the canonical and various crystallographic structures as initial points of departure. All MDs converged on a single dynamical structure of d(ACCGAATTCGGT) in solution. The predicted structure is in close accord with two of the three crystal structures, and indicates that a significant kink in the double helix at the central ApT step in the other crystal molecule may be a packing effect. The dynamical fine structure was analyzed on the basis of helicoidal parameters. The calculated curvature in the sequence was found to originate primarily from YPR steps in the regions flanking the central AATT tract. In order to study the role of structural adaptation of the DNA in the binding process, a subsequent simulation on the 16-mer cognate sequence d(CAACCGAATTCGGTTG) was initiated from the crystallographic coordinates of the bound DNA in the crystal structure of the protein DNA complex. MD simulations starting with the protein-bound form relaxed rapidly back to the dynamical structure predicted from the previous simulations on the uncomplexed DNA. The MD results show that the bound form E2 DNA is a dynamically unstable structure in the absence of protein, and arises as a consequence of both structural changes intrinsic to the sequence and induced by the interaction with protein.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

Abstract

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

Pulling geometry defines the mechanical resistance of a beta-sheet protein

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.     

Molecular dynamics study of time-correlated protein domain motions and molecular flexibility: cytochrome P450BM-3.

Time-correlated atomic motions were used to characterize protein domain boundaries from atomic coordinates generated by molecular dynamics simulations. A novel application of the dynamical cross-correlation matrix (DCCM) analysis tool was used to help identify putative protein domains. In implementing this new approach, several DCCM maps were calculated, each using a different coordinate reference frame from which protein domain boundaries and protein domain residue constituents could be identified. Cytochrome P450BM-3, from Bacillus megaterium, was used as the model protein in this study. The analyses indicated that the simulated protein comprises three distinct domain regions; in contrast, only two protein domains were identified in the original crystal structure report. Specifically, the DCCM analyses showed that the F-G helix region was a separate domain entity and not a part of the alpha domain, as previously designated. The simulations demonstrated that the domain motions of the F-G helix region effected both the size and shape of the enzyme active site, and that the dynamics of the F-G helix domain could possibly control access of substrate to the binding pocket.     

An analysis of the influence of protein intrinsic dynamical properties on its thermal unfolding behavior

The influence of the protein topology-encoded dynamical properties on its thermal unfolding motions was studied in the present work. The intrinsic dynamics of protein topology was obtained by the anisotropic network model (ANM). The ANM has been largely used to investigate protein collective functional motions, but it is not well elucidated if this model can also reveal the preferred large-scale motions during protein unfolding. A small protein barnase is used as a typical case study to explore the relationship between protein topology-encoded dynamics and its unfolding motions. Three thermal unfolding simulations at 500 K were performed for barnase and the entire unfolding trajectories were sampled and partitioned into several windows. For each window, the preferred unfolding motions were investigated by essential dynamics analysis, and then associated with the intrinsic dynamical properties of the starting conformation in this window, which is detected by ANM. The results show that only a few slow normal modes imposed by protein structure are sufficient to give a significant overlap with the preferred unfolding motions. Especially, the large amplitude unfolding movements, which imply that the protein jumps out of a local energy basin, can be well described by a single or several ANM slow modes. Besides the global motions, it is also found that the local residual fluctuations encoded in protein structure are highly correlated with those in the protein unfolding process. Furthermore, we also investigated the relationship between protein intrinsic flexibility and its unfolding events. The results show that the intrinsic flexible regions tend to unfold early. Several early unfolding events can be predicted by analysis of protein structural flexibility. These results imply that protein structure-encoded dynamical properties have significant influences on protein unfolding motions.     

Translational hydration water dynamics drives the protein glass transition

Experimental and computer simulation studies have revealed the presence of a glass-like transition in the internal dynamics of hydrated proteins at approximately 200 K involving an increase of the amplitude of anharmonic dynamics. This increase in flexibility has been correlated with the onset of protein activity. Here, we determine the driving force behind the protein transition by performing molecular dynamics simulations of myoglobin surrounded by a shell of water. A dual heat bath method is used with which, in any given simulation, the protein and solvent are held at different temperatures, and sets of simulations are performed varying the temperature of the components. The results show that the protein transition is driven by a dynamical transition in the hydration water that induces increased fluctuations primarily in side chains in the external regions of the protein. The water transition involves activation of translational diffusion and occurs even in simulations where the protein atoms are held fixed.     

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.     

Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations

The role of Thr729 in modulating the enzymatic function of human topoisomerase I has been characterized by molecular dynamics (MD) simulation. In detail, the structural–dynamical behaviour of the Thr729Lys and the Thr729Pro mutants have been characterized because of their in vivo and in vitro functional properties evidenced in the accompanying paper. Both mutants can bind to the DNA substrate and are enzymatically active, but while Thr729Lys is resistant even at high concentration of the camptothecin (CPT) anti-cancer drug, Thr729Pro shows only a mild reduction in drug sensitivity and in DNA binding. MD simulations show that the Thr729Lys mutation provokes a structural perturbation of the CPT-binding pocket. On the other hand, the Thr729Pro mutant maintains the wild-type structural scaffold, only increasing its rigidity. The simulations also show the complete abolishment, in the Thr729Lys mutant, of the protein communications between the C-terminal domain (where the active Tyr723 is located) and the linker domain, that plays an essential role in the control of the DNA rotation, thus explaining the distributive mode of action displayed by this mutant.     

Mechanically unfolding the small, topologically simple protein L

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.     

Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation

We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule's dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.     

Understanding the structural and dynamic consequences of DNA epigenetic modifications: Computational insights into cytosine methylation and hydroxymethylation

We report a series of molecular dynamics (MD) simulations of up to a microsecond combined simulation time designed to probe epigenetically modified DNA sequences. More specifically, by monitoring the effects of methylation and hydroxymethylation of cytosine in different DNA sequences, we show, for the first time, that DNA epigenetic modifications change the molecule''s dynamical landscape, increasing the propensity of DNA toward different values of twist and/or roll/tilt angles (in relation to the unmodified DNA) at the modification sites. Moreover, both the extent and position of different modifications have significant effects on the amount of structural variation observed. We propose that these conformational differences, which are dependent on the sequence environment, can provide specificity for protein binding.     

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.     

Ultrafast dynamics-driven biomolecular recognition where fast activities dictate slow events

In general, biological macromolecules require significant dynamical freedom to carry out their different functions, including signal transduction, metabolism, catalysis and gene regulation. Effectors (ligands, DNA and external milieu, etc) are considered to function in a purely dynamical manner by selectively stabilizing a specific dynamical state, thereby regulating biological function. In particular, proteins in presence of these effectors can exist in several dynamical states with distinct binding or enzymatic activity. Here, we have reviewed the efficacy of ultrafast fluorescence spectroscopy to monitor the dynamical flexibility of various proteins in presence of different effectors leading to their biological activity. Recent studies demonstrate the potency of a combined approach involving picosecond-resolved Förster resonance energy transfer, polarisation-gated fluorescence and time-dependent stokes shift for the exploration of ultrafast dynamics in biomolecular recognition of various protein molecules. The allosteric protein–protein recognition following differential protein–DNA interaction is shown to be a consequence of some ultrafast segmental motions at the C-terminal of Gal repressor protein dimer with DNA operator sequences O_E and O_I. Differential ultrafast dynamics at the C-terminal of λ-repressor protein with two different operator DNA sequences for the protein–protein interaction with different strengths is also reviewed. We have also systemically briefed the study on the role of ultrafast dynamics of water molecules on the functionality of enzyme proteins α-chymotrypsin and deoxyribonuclease I. The studies on the essential ultrafast dynamics at the active site of the enzyme α-chymotrypsin by using an anthraniloyl fluorescent extrinsic probe covalently attached to the serine-195 residue for the enzymatic activity at homeothermic condition has also been reviewed. Finally, we have highlighted the evidence that a photoinduced dynamical event dictates the molecular recognition of a photochromic ligand, dihydroindolizine with the serine protease α-chymotrypsin and with a liposome (L-α-phosphatidylcholine).     

Dynamical allosterism in the mechanism of action of DNA mismatch repair protein MutS

The multidomain protein Thermus aquaticus MutS and its prokaryotic and eukaryotic homologs recognize DNA replication errors and initiate mismatch repair. MutS actions are fueled by ATP binding and hydrolysis, which modulate its interactions with DNA and other proteins in the mismatch-repair pathway. The DNA binding and ATPase activities are allosterically coupled over a distance of ∼70 Å, and the molecular mechanism of coupling has not been clarified. To address this problem, all-atom molecular dynamics simulations of ∼150 ns including explicit solvent were performed on two key complexes—ATP-bound and ATP-free MutS⋅DNA(+T bulge). We used principal component analysis in fluctuation space to assess ATP ligand-induced changes in MutS structure and dynamics. The molecular dynamics-calculated ensembles of thermally accessible structures showed markedly small differences between the two complexes. However, analysis of the covariance of dynamical fluctuations revealed a number of potentially significant interresidue and interdomain couplings. Moreover, principal component analysis revealed clusters of correlated atomic fluctuations linking the DNA and nucleotide binding sites, especially in the ATP-bound MutS⋅DNA(+T) complex. These results support the idea that allosterism between the nucleotide and DNA binding sites in MutS can occur via ligand-induced changes in motion, i.e., dynamical allosterism.     

Molecular dynamics studies on free and bound targets of the bovine papillomavirus type I e2 protein: the protein binding effect on DNA and the recognition mechanism

Molecular dynamics simulations of a total duration of 30 ns in explicit solvent were carried out on the BPV-1-E2 protein complexed to a high-affinity DNA target containing the two hydrogen-bonded ACCG.CGGT half-sites separated by the noncontacted ACGT sequence. The analysis of the trajectories focuses on the DNA structure and on the dynamics. The data are compared to those issued from recent simulations made on three free targets that recognize E2 with different affinities. E2 does not drastically perturb the mechanic properties of the free DNA: the structural relationships between the BI/BII backbone substates and some helical parameters are preserved in the complex despite a severe slowing down of the phosphate group motions. The structures of both free and bound half-sites are very close to each other although the conformational space explored by these regions is narrowed when they are contacted by the protein. The enhanced plasticity found in the best free target spacers, mainly manifested through the backbone motions, allows a clear overlap between several free and bound global DNA features such as the base displacement. Furthermore, this flexibility is preserved in the complex. Our results support the hypothesis that E2 takes advantage of free predistorted structures that may minimize the DNA deformation cost. In addition, we observe that E2 is far from totally stiffening the DNA, suggesting that the entropic penalty inherent in the complex formation could be limited.     

Toward the understanding of the structure and dynamics of protein-carbohydrate interactions: molecular dynamics studies of the complexes between hevein and oligosaccharidic ligands

Herein we study, through all atom molecular dynamics simulations, the complex between hevein and two N-acetylated chitin oligomers, namely N,N(')-diacetylchitobiose and N,N('),N(")-triacetylchitotriose. The results of the simulations for two disaccharide complexes and one trisaccharide complex show that a carbohydrate oligomer is able to move on the surface of the relatively flat binding pocket of hevein, therefore occupying different binding subpockets. Statistical analysis methods were also applied in order to define the principal overall motions in the complexes, showing how the different ligands in the simulations modulate the protein motions. The oligosaccharide binding can be considered as defined by a subtle balance between enthalpic (formation of intermolecular interactions between the ligand and the receptor) and entropic (due mainly to the possibility for the sugar to move on the surface of the protein domain) effects, determining multiple binding conformations. This structural and dynamical view could parallel the results obtained by regularly used restrained MD simulations based on NOE NMR data that provide a well defined structure for both the disaccharide and trisaccharide complexes, and agrees with the observations for longer oligosaccharide chains.     

Theoretical studies of the response of a protein structure to cavity-creating mutations

We have investigated the response of a protein structure to cavity-creating mutations by molecular dynamics (MD) simulations for the wild-type and the five mutants of phage T4 lysozyme. Essential dynamics (ED) analysis and the methods for calculating different components of local interaction energies are used to examine the structural and energetic characteristics associated with the mutations. In agreement with the x-ray results, it is found that the structural changes due to the replacements of a bulky side chain such as Leu or Phe with Ala within the hydrophobic core can be characterized as slight adjustments rather than substantial reorganization of the protein. The relative stability of different mutant structures can be related with the extent of structural readjustments in response to the mutation. The destabilization of the mutant Leu-->Ala proteins relative to the wild-type is closely related with the loss of van der Waals contacts due to the cavity-creating mutations.