Dynamics Simulations Of Solutions Research Articles

Conformational Plasticity and Druggability of Membrane-Bound K-Ras

Despite decades of studies and the well-established roles of human Ras proteins in health and disease, much remains to be learned about how they work at the molecular level. Major challenges associated with the highly dynamic nature of these proteins and the resolution limits of experimental techniques have hampered our ability to address a number of key issues. These include, at the fundamental level, the sources of signaling specificity and non-overlapping membrane organization of homologous Ras proteins, as well as the structural determinants for the distinct role of specific point mutations in different cancer types. At a more practical level, we lacked the means by which to challenge the “invincibility” of Ras as an anti-cancer drug target. Considerable progress has been made over the last several years toward addressing each of these issues. Molecular simulations and other computational structural biology techniques have made significant contributions to this progress. Our laboratory studies Ras proteins using multi-scale molecular dynamics simulations in solution and membrane environments coupled with a variety of novel concepts, analysis tools and collaborative experiments. The current presentation will focus on the clinically most relevant isoform of Ras proteins: K-Ras. We will discuss our recent efforts toward elucidating the mechanisms of membrane binding and oligomeration of K-Ras as well as the impact of selected point mutations on its dynamics. We will highlight how some of the lessons from Ras can be generalized to other lipid-modified signaling proteins.

In Search of Ras Inhibitors

Membrane binding is essential for the biological activity of Ras proteins, the key regulators of diverse signaling pathways whose malfunction accounts for more than 15% of all human tumors and up to 90% of cases in specific tumor types. However, decades of effort has to date failed to yield selective Ras inhibitors. We hypothesize that disrupting the dynamic coupling between the nucleotide-binding and membrane-interacting regions of Ras by small molecule ligands may have better therapeutic potential. As part of an effort to test this hypothesis, we applied a variety of existing and new ligand binding site identification schemes on ensembles of Ras conformers generated by extensive molecular dynamics simulations in solution and membrane environments. We will discuss the discovery of new pockets that have the potential to bind small molecule ligands, along with alternative strategies for Ras drug design emerging from interactions between various computational and experimental approaches.

Structure and Stability Insights into Tumour Suppressor p53 Evolutionary Related Proteins

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

The nature of the superoxide radical anion in water

Vibrational properties of the superoxide radical anion (O2(-●) in liquid water have been experimentally investigated for the first time. The stretching frequency, its shift from the gas-phase to aqueous solution, anharmonicity constant, and the Raman bandwidths provide an insight into the radical-water interactions and the hydration cage. In view of the spectroscopic information obtained in this work, the structural models based on molecular dynamics simulation in solution and gas-phase infrared studies of the water molecules bound to O2(-●) are critically examined.

In silico characterization of the motor subunit of the e.coli. restriction-modification system EcoR1241

Type1 restriction modification system are intriguing multifunctional multisubunit molecular motors that can catalyze both restriction and modification activity. The type 1 RM enzymes binds to its target sequence and its activity as an endonuclease or methyltransferase is determined by the methylation state of the target sequence. If the target sequence is unmodified, the enzyme while bound to its target site is believed to translocate or pull the DNA towards itself simultaneously in both directions in an ATP dependent manner. The crystal structure of the motor subunit R has been determined by our group but the molecular mechanism by which these enzymes translocate and cleave the DNA is not fully understood. Our current research effort focuses on full-length three-dimensional structures of the R-subunit, utilizing computational and bioinformatics methods. Modelling of missing loops of residues of the crystal structure is done by Modeller9v2. Optimization of intersubunit contacts is performed by energy minimization followed by molecular dynamics simulations in solution at 300K. The dynamic behavior of WT and mutant holo and apo systems is explored by molecular dynamics simulation in GROMACS using the AMBER99SB force field. Conformational changes connected to coupling of translocation and endonuclease activity are observed and QM/MM methods are applied to calculate the contribution of the residue to the overall binding energy of ATP in the binding pocket.

Evaluation of the binding ability of a macrobicyclic receptor for anions by potentiometry and molecular dynamics simulations in solution

Abstract The macrobicycle derived from bis-tren and containing diphenoxy groups as spacers, L1, was synthesized and used as receptor for anions. The binding ability of the new receptor for some aromatic carboxylates [phthalate (ph2−), isophthalate (iph2−), terephthalate (tph2−), benezenetricarboxylate (btc3−), and the herbicide 4-amino-3,5,6-trichloro-2-pyridinecarboxylate (ATCP−)], and the aliphatic cyclohexanetricarboxylate (ch3−) anions was evaluated by potentiometric measurements and molecular dynamic simulation in solution. The association constants were determined by potentiometry in H2O/MeOH (1:1 v/v) at 298.2 K and 0.10 mol dm−3 in KCl. The strongest association was found with btc3− anion and the effective binding constants at pH 5.5 follow the order: btc3−>tph2−>ph2−≈iph2−>ch3−≈ATCP−. Molecular dynamics simulations carried out for the associations of (H6L1)6+ with btc3−, tph2− and iph2− in the same mixture of solvents indicated that these anions interact with the receptor by a combination of electrostatic and multiple N–H⋯O C hydrogen bond interactions. It was also verified that in the recognition process the tph2− remained encapsulated over the entire time of simulation while the btc3− is partially inserted into the receptor cavity with one carboxylate group largely exposed to water solvent molecules, and iph2− anion exhibited an intermediate binding behaviour. The free energy difference between btc3− and iph2− associations estimated by free energy calculations is in excellent agreement with the difference found from the experimental values for the corresponding association constants, which indicates that the unconstrained molecular dynamics simulations carried out with these two anions are realistic pictures of their molecular recognition processes.

IR spectrum of the O–H⋯O hydrogen bond of phthalic acid monomethylester in gas phase and in CCl 4 solution

The absorption spectrum of the title compound in the spectral range of the hydrogen-bonded OH-stretching vibration has been investigated using a five-dimensional gas phase model as well as a QM/MM classical molecular dynamics simulation in solution. The gas phase model predicts a Fermi-resonance between the OH-stretching fundamental and the first OH-bending overtone transition with considerable oscillator strength redistribution. The anharmonic coupling to a low-frequency vibration of the hydrogen bond leading to a vibrational progression is studied within a diabatic potential energy curve model. The condensed phase simulation of the dipole–dipole correlation function results in a broad band in the 3000 cm −1 region in good agreement with experimental data. Further, weaker absorption features around 2600 cm −1 have been identified as being due to motion of the hydrogen within the hydrogen bond.

Network structures in solutions of rigid-chain polyelectrolytes: Computer simulation

The results of molecular dynamics simulation of solutions of highly charged rigid-chain polymers in the presence of multivalent counterions are presented. The processes of self-assembly of macromolecules that occur during the condensation of counterions and depend on temperature, the dielectric permittivity of the medium, and the charge of counterions were studied. Various conformational rearrangements induced by changing the parameters of the medium were examined. The conditions that lead to the formation of network superstructures composed of aggregating polymer chains were found. Size distribution functions for free-volume holes were constructed and the average dimensions of voids in the network structures formed were calculated.

Flexible 5-Guanidino-4-nitroimidazole DNA Lesions: Structures and Thermodynamics

5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring-opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. To elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NI-containing nucleoside with high-level quantum mechanical calculations and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes, with four partner bases opposite the NI. Our results show that NI adopts a planar structure at the damaged base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be nonplanar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. On the basis of hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI-modified duplex contains partner C, consistent with observed incorporation of C opposite NI. However, hydrogen bonding interactions between NI and partner G or A are also found, which would be compatible with the observed mismatches.

Insights into the Structure of Large-Ring Cyclodextrins through Molecular Dynamics Simulations in Solution

Molecular dynamics simulations were carried out using the AMBER parm99 force field and explicit water molecules (TIP3P) to gain insight into the structural deformations and energetics of several large-ring cyclodextrins (with a degree of polymerization 26, 30, 55, 70, 85, and 100) in solution. The structures displayed by CD26 during the MD simulation (10.0 ns) did not correspond to the conformation in the crystalline state. The two "flips" present in the macroring of CD26 in the crystal state disappeared after 1.5 and 3.0 ns simulations, respectively. The larger CDs bear a considerable degree of flexibility. They display different modes of folding and cavity-like regions of different sizes and shapes: circular and elongated loops of variable size, orientated in different fashion; portions of a double helical strand with the two single helices parallel to each other (CD30, CD70); a helix of three turns and a serpentiform portion containing six loops (CD55); a cone-shaped spiral region (CD70); a rounded dendritic fold with several arbitrarily oriented small loops on the surface of the clustering (CD85); three spiral portions and a tendency for bending into two (CD100). These results support the hypothesis for the existence of more than one cavity in large-ring cyclodextrins.

Energetic and entropic contributions to the interactions between like-charged groups in cationic peptides: A molecular dynamics simulation study.

The interaction between like-charged amino acid residues has been proposed to stabilize the folded state of peptides and proteins, and to modulate the substrate binding and the action mechanism of enzymes. We have used an alanine- and lysine-based peptide as a model system to study the interaction between like charges, and we have performed a 16-nsec molecular dynamics simulation in solution. The calculated potential of mean force for the approach of the lysine's Nzeta atoms showed a minimum at a distance of 0.7 nm, in agreement with the separation probabilities obtained from analysis of protein crystal structures. The analysis of the individual energy components showed that the solvent polarization pays for the approach of the like charges and that the van der Waals energies do not contribute significantly. The entropic contributions have been divided in conformational and desolvation terms. Both terms favor the formation of the charge pair. A 10-fold increase in counterion concentration was observed-with respect to its bulk concentration-next to the peptide charges, which helps to stabilize the peptide charges at a close distance.

Supramolecular Chemistry: Computer-Assisted Instruction in Undergraduate and Graduate Chemistry Courses

An interactive electronic textbook (SC-WEB) based on Supramolecular Chemistry, by J.-M. Lehn, is described. It includes the text, which has been specially adapted for teaching, and data on the structures of molecules and supermolecules. The list includes about 100 structures retrieved from the Cambridge Structural Database (version 1997) as well as those obtained from molecular dynamics simulations in solution. The structures can be retrieved either from the text or, independently, from the list.SC-WEB uses two types of programs: the first one reads files in HTML format (Word97, Netscape, or Internet Explorer), and WebLab Viewer Lite is used for visualizing and manipulating the structures. It can run on any PC (W95/NT4.0) computer.The application of SC-WEB for a course in supramolecular chemistry, as well as in other chemical courses, is discussed. A few examples related to the binding of alkali cations by organic and inorganic receptors, and of R-NH3+ cations by organic receptors, are considered in more ...

Molecular dynamics simulations of the Ras:Raf and Rap:Raf complexes

The protein Raf is an immediate downstream target of Ras in the MAP kinase signalling pathway. The complex of Ras with the Ras-binding domain (RBD) of Raf has been modelled by homology to the (E30D,K31E)-Rap1A:RBD complex, and both have been subjected to multiple molecular dynamics simulations in solution. While both complexes are stable, several rearrangements occur in the Ras:RBD simulations: the RBD loop 100-109 moves closer to Ras, Arg73 in the RBD moves towards Ras to form a salt bridge with Ras-Asp33, and Loop 4 of the Ras switch II region shifts upwards toward the RBD. The Ras:RBD interactions (including the RBD-Arg73 interaction) are consistent with available NMR and mutagenesis data on the Ras: RBD complex in solution. The Ras switch II region does not interact directly with the RBD, although indirect interactions exist through the effector domain and bridging water molecules. No large-scale RBD motion is seen in the Ras:RBD complex, compared to the Rap:RBD complex, to suggest an allosteric activation of Raf by Ras. This may be because the Raf kinase domain (whose structure is unknown) is not included in the model.

Structure, dynamics and hydration of the nogalamycin-d(ATGCAT)2 complex determined by NMR and molecular dynamics simulations in solution

The structure of the 1:1 nogalamycin:d(ATGCAT)2 complex has been determined in solution from high-resolution NMR data and restrained molecular dynamics (rMD) simulations using an explicit solvation model. The antibiotic intercalates at the 5′-TpG step with the nogalose lying along the minor groove towards the centre of the duplex. Many drug-DNA nuclear Overhauser enhancements (NOEs) in the minor groove are indicative of hydrophobic interactions over the TGCA sequence. Steric occlusion prevents a second nogalamycin molecule from binding at the symmetry-related 5′-CpA site, leading to the conclusion that the observed binding orientation in this complex is the preferred orientation free of the complication of end-effects (drug molecules occupy terminal intercalation sites in all X-ray structures) or steric interactions between drug molecules (other NMR structures have two drug molecules bound in close proximity), as previously suggested. Fluctuations in key structural parameters such as rise, helical twist, slide, shift, buckle and sugar pucker have been examined from an analysis of the final 500 ps of a 1 ns rMD simulation, and reveal that many sequence-dependent structural features previously identified by comparison of different X-ray structures lie within the range of dynamic fluctuations observed in the MD simulations. Water density calculations on MD simulation data reveal a time-averaged pattern of hydration in both the major and minor groove, in good agreement with the extensive hydration observed in two related X-ray structures in which nogalamycin is bound at terminal 5′-TpG sites. However, the pattern of hydration determined from the sign and magnitude of NOE and ROE cross-peaks to water identified in 2D NOESY and ROESY experiments identifies only a few “bound” water molecules with long residence times. These solvate the charged bicycloaminoglucose sugar ring, suggesting an important role for water molecules in mediating drug-DNA electrostatic interactions within the major groove. The high density of water molecules found in the minor groove in X-ray structures and MD simulations is found to be associated with only weakly bound solvent in solution.

Molecular Dynamics Simulation of Colchicinoids

Colchicine, a tricyclic alkaloid, has a remarkable range of biological activities. It binds with tubulin and prevents the formation of microtubules. This compound consists of a six membered aromatic ring (A ring), a seven membered troponoid ring (C ring) and another seven membered aliphatic ring (B ring). Using molecular mechanics and molecular dynamics simulations as tools, conformational analysis of colchicine and its several important analogs were done. Molecular mechanics studies show that conformational space of these molecules have one low energy region. Taking the low energy minima as the starting conformation, molecular dynamics simulation for 100 pico seconds is done for each of the analogs and molecular dynamics simulation in solution is done for three representative compounds colchicine, isocolchicine and A-C compound. Internal coordinate trajectories show that the value of the dihedral angle C9-C7-C1-C14 (phi), (C7-C1 bond connects the A and C ring), is within 40° to 50° for all the compounds with fluctuations less than 15°. These calculations indicate that there is an overall similarity in the dynamically averaged structure of all the drugs. The A ring and B ring of the compounds are more or less rigid. The C ring is somewhat flexible, the average conformation and motional properties show overall similarity. The potential energy curve and dynamics behaviour of colchicine and isocolchicine suggests that the difference in binding property of colchine and isocolchicine may originate from the positional difference of carbonyl oxygen and methoxy group of C ring, which is the only difference in the structures of the two compounds and this has no effect on the motional property and average conformations of these two compounds. From our study it is proposed that the movements occuring at various positions of the drug molecules are significantly correlated. It is suggested that such correlated motion may play an important role in the biological property of these compounds.

DYNAX: A New System for Biomolecular Dynamics Simulation in Solution

DYNAX: A New System for Biomolecular Dynamics Simulation in Solution

Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin.

Molecular dynamics simulations in solution are performed for a rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus (RdPf) and one from the mesophilic organism Desulfovibrio vulgaris (RdDv). The two proteins are simulated at four temperatures: 300 K, 373 K, 473 K (two sets), and 500 K; the various simulations extended from 200 ps to 1,020 ps. At room temperature, the two proteins are stable, remain close to the crystal structure, and exhibit similar dynamic behavior; the RMS residue fluctuations are slightly smaller in the hyperthermophilic protein. An analysis of the average energy contributions in the two proteins is made; the results suggest that the intraprotein energy stabilizes RdPf relative to RdDv. At 373 K, the mesophilic protein unfolds rapidly (it begins to unfold at 300 ps), whereas the hyperthermophilic does not unfold over the simulation of 600 ps. This is in accord with the expected stability of the two proteins. At 473 K, where both proteins are expected to be unstable, unfolding behavior is observed within 200 ps and the mesophilic protein unfolds faster than the hyperthermophilic one. At 500 K, both proteins unfold; the hyperthermophilic protein does so faster than the mesophilic protein. The unfolding behavior for the two proteins is found to be very similar. Although the exact order of events differs from one trajectory to another, both proteins unfold first by opening of the loop region to expose the hydrophobic core. This is followed by unzipping of the beta-sheet. The results obtained in the simulation are discussed in terms of the factors involved in flexibility and thermostability.

Molecular dynamics simulation of the energy distributions of molecules in liquid solutions

Abstract A solution is modelled as a classical 2D molecular system consisting of two types of molecules having different interaction parameters. The interaction is modelled by Lennard - Jones - type potentials. Molecular Dynamics simulations of solutions of soft molecules imbedded into a bath of rather hard solvent molecules are performed. From the molecular dynamics trajectories we derive the energy distributions and study local energy excitations of the solute and the solvent molecules. In particular the dynamics of activation processes (high energy events) is investigated for a special model: We simulate the thermal equilibrium of one molecule with Lennard-Jones 8-6-interactions imbedded into a bath of molecules with 18-6 interactions. It is shown that in thermal equilibrium a region of temperatures and densities exists, where the mean one-particle energy has a relative maximum. In this region the potential part of the thermal energy is preferably partitioned to the solvent molecules. Further we show that the energy distribution of the soft molecules shows longer tails than that of the hard molecules. On the basis of these distributions it is concluded that energy spots at the solute molecules may appear which possibly could lead to the enhancement of solute reactions.

Selectin-ligand interactions revealed by molecular dynamics simulation in solution.

Through a computer modeling and simulation technique, we investigated the binding mode of a complex of E-selectin-GSC-150, which is a novel selectin blocker. GSC-150 is the 3'-sulfated Lewis X derivative with a long, branched alkyl chain. Initial attempts to construct a model for E-selectin-GSC-150 complex were performed based on a previously reported model of E-selectin-sialyl Lewis X (sLex) complex [Kogan, T.P.; Revelle, B.M.; Tapp, S.; Scott, D.; Beck, P.J.J. Biol. Chem. 1995, 270, 14047-14055]. In our model, the carbohydrate portion of GSC-150 interacted with the protein in a similar manner as that of sLex reported previously. Interestingly, each of the branched alkyl chains extended on the surface of E-selectin and interacted with two different hydrophobic portions. One of these hydrophobic portions consists of Tyr44, Pro46, and Tyr48. Another portion forms a shallow cavity, and it consists of Ala9, Leu114, and the alkyl moieties of the side chains of Lys111, Lys112, and Lys113. A subsequent 200-ps molecular dynamics simulation in solution revealed that the interactions involved in the sugar portion of the ligand were relatively weak, whereas the hydrophobic interactions involved in the branched alkyl chains were fairly stable in solution. These results suggest that the branched alkyl chain serves as an "anchor" for the tight binding of GSC-150 on the surface of E-Selectin. This is the first attempt to evaluate the dynamics of E-Selectin-ligand interactions in solution, and it sheds light on the nature of ligand recognition by selectins.

Site-Directed Mutation Study on Hyperthermostability of Rubredoxin from Pyrococcus furiosus Using Molecular Dynamics Simulations in Solution

The hyperthermostable protein rubredoxin from Pyrococcus furiosus is a 53-residue protein with a three-stranded antiparallel β-sheet and several loops. In this paper, the hydrophobic interaction of residues on the surface of the protein was investigated as well as electrostatic interactions between residues. To investigate the effect of changes of electrostatic and hydrophobic interactions on the structure and the dynamic property of P. furiosus rebredoxin, molecular dynamics simulations in solution were performed on three mesophilic rubredoxins, P. furiosus rubredoxin, and five mutants of P. furiosus rubredoxin. Glu 14 of P. furiosus has a backbone hydrogen bond with N-terminal and multiple electrostatic interactions with Ala 1, Trp 3, and Phe 29. The multiple electrostatic interactions make the residues around the N-terminal stable, and the hydrogen bond between Glu 14 and Ala 1 remains even at high temperature. The flexibility of a loop from Asp 15 to Gly 26 is reduced by making the loop closer to the ...