The consistency of large concerted motions in proteins in molecular dynamics simulations.

A detailed investigation is presented into the effect of limited sampling time and small changes in the force field on molecular dynamics simulations of a protein. Thirteen independent simulations of the B1 IgG-binding domain of streptococcal protein G were performed, with small changes in the simulation parameters in each simulation. Parameters studied included temperature, bond constraints, cut-off radius for electrostatic interactions, and initial placement of hydrogen atoms. The essential dynamics technique was used to reveal dynamic differences between the simulations. Similar essential dynamics properties were found for all simulations, indicating that the large concerted motions found in the simulations are not particularly sensitive to small changes in the force field. A thorough investigation into the stability of the essential dynamics properties as derived from a molecular dynamics simulation of a few hundred picoseconds is provided. Although the definition of the essential modes of motion has not fully converged in these short simulations, the subspace in which these modes are confined is found to be reproducible.     

Conformational changes in bacteriophage phi 29 connector prevents DNA-binding activity

In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.     

Homology modelling and molecular dynamics simulations: comparative studies of human aquaporin-1

The structures of the mammalian water transport protein Aqp1 and of its bacterial homologue GlpF enables us to test whether homology models can be used to explore relationships between structure, dynamics and function in mammalian transport proteins. Molecular dynamics simulations (totalling almost 40 ns) were performed starting from: the X-ray structure of Aqp1; a homology model of Aqp1 based on the GlpF structure; and intermediate resolution structures of Aqp1 derived from electron microscopy. Comparisons of protein RMSDs vs. time suggest that the homology models are of comparable conformational stability to the X-ray structure, whereas the intermediate resolution structures exhibit significant conformation drift. For simulations based on the X-ray structure and on homology models, the flexibility profile vs. residue number correlates well with the crystallographic B-values for each residue. In the simulations based on intermediate resolution structures, mobility of the highly conserved NPA loops is substantially higher than in the simulations based on the X-ray structure or the homology models. Pore radius profiles remained relatively constant in the X-ray and homology model simulations but showed substantial fluctuations (reflecting the higher NPA loop mobility) in the intermediate resolution simulations. The orientation of the dipoles of water molecules within the pore is of key importance in maintaining low proton permeability through Aqp1. This property seems to be quite robust to the starting model used in the simulation. These simulations suggest that homology models based on bacterial homologues may be used to derive functionally relevant information on the structural dynamics of mammalian transport proteins.     

Orthobaric Densities from Simulations of the Liquid Vapour Interface

Molecular dynamics studies of the liquid vapour interface of a Lennard-Jones fluid performed on a CYBER 205 are reported for kT/ε = 0.70, 0.85, and 1.10. The main emphasis is on the orthobaric fluid densities: their dependence on different cut-off radii and long range corrections as well as their comparison with the coexisting densities from bulk fluid simulations. As previously reported bubble densities from bulk simulations differ by about 3 per cent at kT/ε = 1.10, phase equilibrium was redetermined there with the NpT + test particle method. Concerning the bubble densities from the interface simulations, increase of the cut-off radius and use of long range corrections shift them up rather close to the “bulk” values. The dew densities from the interface simulations which for the usual cut-off radius of 2.5 [sgrave] are mostly too high by a factor of 3 are decreased dramatically by accounting for the long range part of the potential. For a cut-off radius of 5 [sgrave] and long range corrections they agree with the “bulk” values within the accuracy of the latters which, however, is estimated to be only by about 20 per cent. Some characteristic density profiles are also shown.     

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.     

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.     

Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis-induced conformational change

Members of a superfamily of RecA-like recombinases facilitate a central strand exchange reaction in the DNA repair process. Archaeal RadA and Rad51 and eukaryal Rad51 and meiosis-specific DMC1 form a closely related group of recombinases distinct from bacterial RecA. Nevertheless, all such recombinases share a conserved core domain which carries the ATPase site and putative DNA-binding sites. Here we present the crystal structure of an archaeal RadA from Methanococcus voltae (MvRadA) in complex with ADP and Mg2+ at 2.1 A resolution. The crystallized RadA-ADP filament has an extended helical pitch similar to those of previously determined structures in the presence of nonhydrolyzable ATP analogue AMP-PNP. Structural comparison reveals two recurrent conformations with an extensive allosteric effect spanning the ATPase site and the putative DNA-binding L2 region. Varied conformations of the L2 region also imply a dynamic nature of recombinase-bound DNA.     

Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA

The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8-10 nucleotides (nt)] and stable ( approximately 30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and flanked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix-turn-helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8-10 to 30 nt mode is mediated by DNA binding with the trimerization core.     

Identification of core allosteric sites through temperature- and nucleus-invariant chemical shift covariance

Allosteric regulation is essential to control biological function. In addition, allosteric sites offer a promising venue for selective drug targeting. However, accurate mapping of allosteric sites remains challenging since allostery relies on often subtle, yet functionally relevant, structural and dynamical changes. A viable approach proposed to overcome such challenge is chemical shift covariance analysis (CHESCA). Although CHESCA offers an exhaustive map of allosteric networks, it is critical to define the core allosteric sites to be prioritized in subsequent functional studies or in the design of allosteric drugs. Here, we propose two new CHESCA-based methodologies, called temperature CHESCA (T-CHESCA) and CLASS-CHESCA, aimed at narrowing down allosteric maps to the core allosteric residues. Both T- and CLASS-CHESCAs rely on the invariance of core inter-residue correlations to changes in the chemical shifts of the active and inactive conformations interconverting in fast exchange. In T-CHESCA the chemical shifts of such states are modulated through temperature changes, while in CLASS-CHESCA through variations in the spin-active nuclei involved in pairwise correlations. T- and CLASS-CHESCAs, as well as complete-linkage CHESCA, were applied to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC), which serves as a prototypical allosteric switch. Residues consistently identified by the three CHESCA methods were found in previously identified EPAC allosteric core sites. Hence, T-, CLASS-, and CL-CHESCA provide a toolset to establish allosteric site hierarchy and triage allosteric sites to be further analyzed by mutations and functional assays. Furthermore, the core allosteric networks selectively revealed through T- and CLASS-CHESCA are expected to facilitate the mechanistic understanding of disease-related mutations and the design of selective allosteric modulators.     

The structure of an HIV-1 specific cell entry inhibitor in complex with the HIV-1 gp41 trimeric core

The three-dimensional structure of the complex between an HIV-1 cell-entry inhibitor selected from screening a combinatorial library of non-natural building blocks and the central, trimeric, coiled-coil core of HIV-1 gp41 has been determined by X-ray crystallography. The biased combinatorial library was designed to identify ligands binding in nonpolar pockets on the surface of the coiled-coil core of gp41. The crystal structure shows that the non-peptide moiety of the inhibitor binds to the targeted cavity in two different binding modes. This result suggests a strategy for increasing inhibitor potency by use of a second-generation combinatorial library designed to give simultaneous occupancy of both binding sites.     

Structure and dynamics of calmodulin in solution.

To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides.     

Theoretical studies of relaxation of a monomeric subunit of HIV-1 protease in water using molecular dynamics

The dynamic behavior of one 99-residue subunit of the dimeric aspartyl protease of HIV-1 was studied in a 160 psec molecular dynamics simulation at 300 K in water. The crystal structure of one of the identical subunits of the dimer was the starting point, with the aqueous phase modeled by 4,331 explicit waters in a restrained spherical droplet Analysis of the simulations showed that the monomer displayed considerable flexibility in the interfacial portions of the flap (the region which folds over the substrate), the N- and C-0termini, and, to a lesser extent, the active site. The flap undergoes significant motion as an independent rigid finger, but without the cantilever previously reported hi a simulation of the dimer. The N-terminus displayed the greatest fluctuational disorder whereas the C-terminus exhibited the greatest root mean square movement from the crystal structure. The central core of the monomer had a heavy-atom root mean square deviation from the initial structure of about 3.0 Å during the latter half of the simulation. Although this is larger than the 1.6 Å found for comparable simulations of typical globular proteins, the general features of the tertiary structure were preserved over the course of the simulation. Overall, these results indicate that the relaxed structure obtained in these simulations may provide a better model for the tertiary structure of the solvated HIV-1 protease monomer than the subunit conformation seen in the X-ray crystallographic structure of the dimer. Except in the flap region, the design of compounds intended to interfere with dimerization should take this relaxation and the flexibility of the solvated monomer, especially at the termini, into account. © 1993 Wiley-Liss, Inc.     

Crystal structure of the DNA-binding domain of BldD, a central regulator of aerial mycelium formation in Streptomyces coelicolor A3(2)

BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 angstroms resolution structure of the DNA-binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (alpha1-alpha4) containing a helix-turn-helix motif (alpha2-alpha3) resembling that of the DNA-binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of alpha3 and the 'turn' region between alpha2 and alpha3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first-identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.     

Behavior of BsoBI endonuclease in the presence and absence of DNA

BsoBI is a type II restriction endonuclease belonging to the EcoRI family. There is only one previously published X-ray structure for this endonuclease: it shows a homodimer of BsoBI completely encircling DNA in a tunnel. In this work, molecular dynamics simulations were employed to elucidate possible ways in which DNA is loaded into this complex prior to its cleavage. We found that the dimer does not open spontaneously when DNA is removed from the complex on the timescale of our simulations (~ 0.5 μs). A biased simulation had to be used to facilitate the opening, which revealed a possible way for the two catalytic domains to separate. The α-helices connecting the catalytic and helical domains were found to act as a hinge during the separation. In addition, we found that the opening of the BsoBI dimer was influenced by the type of counterions present in the environment. A reference simulation of the BsoBI/DNA complex further showed spontaneous reorganization of the active sites due to the binding of solvent ions, which led to an active-site structure consistent with other experimental structures of type II restriction endonucleases determined in the presence of metal ions.     

Monte Carlo kilovoltage X-ray tube simulation: A statistical analysis and compact simulation method

IntroductionMonte Carlo (MC) simulations are a powerful tool for improving image quality in X-ray based imaging modalities. An accurate X-ray source model is essential to MC modeling for CBCT but can be difficult to implement on a GPU while maintaining efficiency and memory limitations. A statistical analysis of the photon distribution from a MC X-ray tube simulation is conducted in hopes of building a compact source model.Materials & methodsMC simulations of an X-ray tube were carried out using BEAMnrc. The resulting photons were sorted into four categories: primary, scatter, off-focal radiation (OFR), and both (scatter and OFR). A statistical analysis of the photon components (energy, position, direction) was completed. A novel method for a compact (memory efficient) representation of the PHSP data was implemented and tested using different statistical based linear transformations (PCA, ZCA, ICA), as well as a geometrical transformation.ResultsThe statistical analysis showed all photon groupings had strong correlations between position and direction, with the largest correlation in the primary data. The novel method was successful in compactly representing the primary (error < 2%) and scatter (error < 6%) photon groupings by reducing the component correlations.Discussion & conclusionStatistical linear transforms provide a method of reducing the memory required to accurately simulate an X-ray source in a GPU MC system. If all photon types are required, the proposed method reduces the memory requirements by 3.8 times. When only primary and scatter data is needed, the memory requirement is reduced from gigabytes to kilobytes.     

A comparison between two prokaryotic potassium channels (KirBac1.1 and KcsA) in a molecular dynamics (MD) simulation study

The two potassium ion channels KirBac1.1 and KcsA are compared in a Molecular Dynamics (MD) simulation study. The location and motion of the potassium ions observed in the simulations are compared to those in the X-ray structures and previous simulations. In our simulations several of the crystallography resolved ion sites in KirBac1.1 are occupied by ions. In addition to this, two in KirBac1.1 unresolved sites where occupied by ions at sites that are in close correspondence to sites found in KcsA. There is every reason to believe that the conserved alignment of the selectivity filter in the potassium ion channel family corresponds to a very similar mechanism for ion transport across the filter. The gate residues, Phe146 in KirBac1.1 and Ala111 in KcsA acted in the simulations as effective barriers which never were passed by ions nor water molecules.     

Molecular dynamics studies of a DNA-binding protein: 1. A comparison of the trp repressor and trp aporepressor aqueous simulations.

The results of two 30-ps molecular dynamics simulations of the trp repressor and trp aporepressor proteins are presented in this paper. The simulations were obtained using the AMBER molecular mechanical force field and in both simulations a 6-A shell of TIP3P waters surrounded the proteins. The trp repressor protein is a DNA-binding regulatory protein and it utilizes a helix-turn-helix (D helix-turn-E helix) motif to interact with DNA. The trp aporepressor, lacking two molecules of the L-tryptophan corepressor, cannot bind specifically to DNA. Our simulations show that the N- and C-termini and the residues in and near the helix-turn-helix motifs are the most mobile regions of the proteins, in agreement with the X-ray crystallographic studies. Our simulations also find increased mobility of the residues in the turn-D helix-turn regions of the proteins. We find the average distance separating the DNA-binding motifs to be larger in the repressor as compared to the aporepressor. In addition to examining the protein residue fluctuations and deviations with respect to X-ray structures, we have also focused on backbone dihedral angles and corepressor hydrogen-bonding patterns in this paper.