Dynamics Simulations In Implicit Solvent Research Articles

Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics.

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.

Exhaustive Exploration of the Conformational Landscape of Small Cyclic Peptides Using a Robotics Approach.

Small cyclic peptides represent a promising class of therapeutic molecules with unique chemical properties. However, the poor knowledge of their structural characteristics makes their computational design and structure prediction a real challenge. In order to better describe their conformational space, we developed a method, named EGSCyP, for the exhaustive exploration of the energy landscape of small head-to-tail cyclic peptides. The method can be summarized by (i) a global exploration of the conformational space based on a mechanistic representation of the peptide and the use of robotics-based algorithms to deal with the closure constraint and (ii) an all-atom refinement of the obtained conformations. EGSCyP can handle D-form residues and N-methylations. Two strategies for the side-chains placement were implemented and compared. To validate our approach, we applied it to a set of three variants of cyclic RGDFV pentapeptides, including the drug candidate Cilengitide. A comparative analysis was made with respect to replica exchange molecular dynamics simulations in implicit solvent. Its results show that the EGSCyP method provides a very complete characterization of the conformational space of small cyclic pentapeptides.

Protein structure model refinement in CASP12 using short and long molecular dynamics simulations in implicit solvent.

Protein structure prediction has matured over years, particularly those which use structure templates for building a model. It can build a model with correct overall conformation in cases where appropriate templates are available. Models with the correct topology can be practically useful for limited purposes that need residue-level accuracy, but further improvement of the models can allow the models to be used in tasks that need detailed structures, such as molecular replacement in X-ray crystallography or structure-based drug screening. Thus, model refinement is an important final step in protein structure prediction to bridge predictions to real-life applications. Model refinement is one of the categories in recent rounds of critical assessment of techniques in protein structure prediction (CASP) and has recently been drawing more attention due to its realized importance. Here we report our group's performance in the refinement category in CASP12. Our method is based on inexpensive short molecular dynamics (MD) simulations in implicit solvent. Our performance in CASP12 was among the top, which was consistent with the previous round, CASP11. Our method with short MD runs achieved comparable performance with other methods that used longer simulations. Detailed analyses found that improvements typically occurred in entire regions of a structure rather than only in flexible loop regions. The remaining challenge in the structure refinement includes large conformational refinement which involves substantial motions of secondary structure elements or domains.

Characterization of the Interactions of Arg-Gly-Asp- and Ala-Gly-Asp-Val-Containing Peptides with the Platelet Integrin αIIbβ3

Binding of soluble fibrinogen to the activated open conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. Paradoxically, however, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3. Moreover, the Aα chain RGD motifs make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. The interaction of αIIbβ3 with a variety of RGD- and γC-12 peptides has been studied extensively, but the characteristics of their binding to the active open and inactive closed conformations of αIIbβ3 are largely unknown. Here, we have used both experimental and computational approaches to compare the two-dimensional kinetics, thermodynamics and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. First, we employed optical trap-based single-molecule nanomechanical measurements to determine the probability of peptide binding to αIIbβ3 as a function of the time the peptides interact with αIIbβ3. In the optical trap-based experimental system, a microscopic bead coated with either cRGDFK or γC-12 is trapped in a fluid chamber by a focused laser beam and moved in an oscillatory manner to touch a stationary pedestal coated with αIIbβ3. When the peptide on the bead interacts with αIIbβ3 on the pedestal, tension is generated when the bead is displaced from the laser focus until the αIIbβ3-peptide bond ruptures. The percentage of binding/unbinding events at a particular time of contact duration enabled us to plot the force-free binding probability as a function of contact duration. From these curves, we extracted first-order binding rates: 0.61×10-14 cm2/s for γC-12 and 0.71×10-14 cm2/s for cRGDFK and unbinding rates: 2.05/s and 1.53/s for the γC-12 and cRGDFK, respectively. Corresponding binding affinity constants were 0.3×10-14 cm2 and 0.46×10-14 cm2 for the γC-12 and cRGDFK, respectively, indicating that cRGDFK binds to αIIbβ3 somewhat tighter than γC-12. These measurements were then followed by computational modeling using NMR-determined 3D solution structures of cRGDFK and γC-12, as well as the crystallographically resolved αIIbβ3 conformers of the αIIbβ3 headpiece (PDB entries: 3ZDX and 3ZE2). Docking analysis confirmed that both cRGDFK and γC-12 bound to the αIIbβ3 conformers at a site located in the interface between the α subunit β-propeller domain and the β subunit βI domain. Estimated docking energies revealed that the closed αIIbβ3 conformation is almost as reactive towards the peptides as the open conformation. Next, we probed the strength of the αIIbβ3-peptide interactions and determined their comparative binding energies and the dynamics of interatomic contacts by performing dynamic force-ramp unbinding in silico using all-atom Molecular Dynamics simulations in implicit solvent on Graphics Processing Units. Force was increased linearly at a pulling rate of 2×104 μm/s and applied to specified Cα-atom of the peptides. Analysis of the molecular force profiles and the number of ligand-receptor binding contacts revealed that both peptides interact strongly with αIIbβ3, forming stable bimolecular complexes that dissociate in the 60-120 pN range through several competing unbinding pathways that display multi-step kinetics and distinct bond lifetimes. We profiled the Gibbs free energy (ΔG) of the αIIbβ3-peptide complexes as a function of the separation distance between the open form of αIIbβ3 headpiece and the peptide using the Umbrella Sampling technique. The results showed that the energies of ligand binding to the αIIbβ3 headpiece are ΔG~16 kcal/mol for cRGDFK and ΔG~13 kcal/mol for γC-12, meaning that the overall thermodynamic stability of the αIIbβ3-peptide complex is higher for cRGDFK than for γC-12. Thus, these results provide a kinetic and thermodynamic explanation for previous observations that the affinity of RGD for αIIbβ3 is greater than AGDV and support our hypothesis that the RGD motifs preferentially support the interaction of αIIbβ3 with immobilized fibrinogen and fibrin. DisclosuresWeisel:Bayer: Research Funding.

Inhibition of the ferric uptake regulator by peptides derived from anti-FUR peptide aptamers: coupled theoretical and experimental approaches.

The FUR protein (ferric uptake regulator) is an iron-dependent global transcriptional regulator. Specific to bacteria, FUR is an attractive antibacterial target since virulence is correlated to iron bioavailability. Recently, four anti-FUR peptide aptamers, composed of 13 amino acid variable loops inserted into a thioredoxinA scaffold, were identified, which were able to interact with Escherichia coli FUR (EcFUR), inhibit its binding to DNA and to decrease the virulence of pathogenic E. coli in a fly infection model. The first characterization of anti-FUR linear peptides (pF1 6 to 13 amino acids) derived from the variable part of the F1 anti-FUR peptide aptamer is described herein. Theoretical and experimental approaches, in original combination, were used to study interactions of these peptides with FUR in order to understand their mechanism of inhibition. After modeling EcFUR by homology, docking with Autodock was combined with molecular dynamics simulations in implicit solvent to take into account the flexibility of the partners. All calculations were cross-checked either with other programs or with experimental data. As a result, reliable structures of EcFUR and its complex with pF1 are given and an inhibition pocket formed by the groove between the two FUR subunits is proposed. The location of the pocket was validated through experimental mutation of key EcFUR residues at the site of proposed peptide interaction. Cyclisation of pF1, mimicking the peptide constraint in F1, improved inhibition. The details of the interactions between peptide and protein were analyzed and a mechanism of inhibition of these anti-FUR molecules is proposed.

Mechanical Probes of SOD1 Predict Systematic Trends in Metal and Dimer Affinity of ALS-Associated Mutants

Mutations and oxidative modification in the protein Cu,Zn superoxide dismutase (SOD1) have been implicated in the death of motor neurons in amyotrophic lateral sclerosis (ALS), a presently incurable, invariably fatal neurodegenerative disease. Here we employ steered, all-atom molecular dynamics simulations in implicit solvent to investigate the significance of either mutations or post-translational modifications (PTMs) to SOD1 on metal affinity, dimer stability, and mechanical malleability. The work required to induce moderate structural deformations as a function of sequence index constitutes a “mechanical fingerprint” measuring structural rigidity in the native basin, from which we are able to unambiguously distinguish wild-type SOD1 from PTM variants and measure the severity of a given PTM on structural integrity. The cumulative distribution of work values provided a way to cleanly discriminate between SOD1 variants. Disulfide reduction destabilizes dimer stability more than the removal of either metal, but not moreso than the removal of both metals. Intriguingly, we found that disulfide reduction mechanically stabilizes apo-SOD1 monomer, underscoring the differences between native basin mechanical properties and equilibrium thermodynamic stabilities and elucidating the presence of internal stress in the apo state. All PTMs and ALS-associated mutants studied showed an increased tendency to lose either Cu or Zn and to monomerize—processes known to be critical in the progression of ALS. The valence of Cu strongly modulates its binding free energy. As well, several mutants were more susceptible to loss of metals and monomerization than the disulfide-reduced or apo forms of SOD1. Distance constraints are required to calculate free energies for metal binding and dimer separation, which are validated using thermodynamic cycles. When distance constraints are removed, the results agree with those obtained from direct application of the Jarzynski equality.

Эффективные генераторы псевдослучайных чисел при молекулярном моделировании на видеокартах

Динамика Ланжевена, метод Монте-Карло и моделирование молекулярной динамики в неявном растворителе требуют больших массивов случайных чисел на каждом шаге расчета. Мы исследовали два подхода в реализации генераторов на графических процессорах. Первый реализует последовательный алгоритм генератора на каждом потоке в отдельности. Второй основан на возможности взаимодействия между потоками и реализует общий алгоритм на всех потоках в целом. Мы покажем использование этих подходов на примере алгоритмов Ran 2, Hybrid Taus и Lagged Fibonacci. Для проверки случайности полученных чисел мы использовали разработанные генераторы при моделировании динамики Ланжевена N независимых гармонических осцилляторов в термостате. Это позволило нам оценить статистические характеристики генераторов. Мы также исследовали производительность, использование памяти и ускорение, получаемое при переносе алгоритма с центрального на графический процессор.

A Repulsive Electrostatic Mechanism for Protein Export through the Type III Secretion Apparatus

Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus, which is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. The trajectories reveal a screwlike rotation motion during the export of nativelike helix-turn-helix conformations. Interestingly, the channel interior with excessive electronegative potential creates an energy barrier for MxiH to enter the channel, whereas the same may facilitate the ejection of the effectors into host cells. Structurally known basal regions and ATPase underneath the basal region also have electronegative interiors. Effector proteins also have considerable electronegative potential patches on their surfaces. From these observations, we propose a repulsive electrostatic mechanism for protein translocation through the type III secretion apparatus. Based on this mechanism, the ATPase activity and/or proton motive force could be used to energize the protein translocation through these nanomachines. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

Molecular Simulation of ab Initio Protein Folding for a Millisecond Folder NTL9(1−39)

To date, the slowest-folding proteins folded ab initio by all-atom molecular dynamics simulations have had folding times in the range of nanoseconds to microseconds. We report simulations of several folding trajectories of NTL9(1-39), a protein which has a folding time of approximately 1.5 ms. Distributed molecular dynamics simulations in implicit solvent on GPU processors were used to generate ensembles of trajectories out to approximately 40 micros for several temperatures and starting states. At a temperature less than the melting point of the force field, we observe a small number of productive folding events, consistent with predictions from a model of parallel uncoupled two-state simulations. The posterior distribution of the folding rate predicted from the data agrees well with the experimental folding rate (approximately 640/s). Markov State Models (MSMs) built from the data show a gap in the implied time scales indicative of two-state folding and heterogeneous pathways connecting diffuse mesoscopic substates. Structural analysis of the 14 out of 2000 macrostates transited by the top 10 folding pathways reveals that native-like pairing between strands 1 and 2 only occurs for macrostates with p(fold) > 0.5, suggesting beta(12) hairpin formation may be rate-limiting. We believe that using simulation data such as these to seed adaptive resampling simulations will be a promising new method for achieving statistically converged descriptions of folding landscapes at longer time scales than ever before.

Binding Pathways of Ligands to HIV‐1 Protease: Coarse‐grained and Atomistic Simulations

Multiscale simulations (coarse-grained Brownian dynamics simulations and all-atom molecular dynamics simulations in implicit solvent) were applied to reveal the binding processes of ligands as they enter the binding site of the HIV-1 protease. The initial structures used for the molecular dynamics simulations were generated based on the Brownian dynamics trajectories, and this is the first molecular dynamics simulation of modeling the association of a ligand with the protease. We found that a protease substrate successfully binds to the protein when the flaps are fully open. Surprisingly, a smaller cyclic urea inhibitor (XK263) can reach the binding site when the flaps are not fully open. However, if the flaps are nearly closed, the inhibitor must rearrange or binding can fail because the inhibitor cannot attain proper conformations to enter the binding site. Both the peptide substrate and XK263 can also affect the protein's internal motion, which may help the flaps to open. Simulations allow us to efficiently study the ligand binding processes and may help those who study drug discovery to find optimal association pathways and to design those ligands with the best binding kinetics.

Structure and Interactions of the Ubiquitin-Conjugating Enzyme Variant Human Uev1a: Implications for Enzymatic Synthesis of Polyubiquitin Chains,

Lys(63)-linked polyubiquitination of TRAF2 or TRAF6 is an essential step within the signal transduction cascade responsible for activation of p38, c-Jun N-terminal kinase, and the transcription factor NF-kappaB. Attachment of ubiquitin (Ub) to a TRAF, and conjugation of Ub molecules to form a polyUb chain, is catalyzed by a heterodimer composed of a catalytically active E2 (hUbc13), involved in covalent bond transfer, and hUev1a, an E2-like protein involved in substrate Ub binding. Given the key biochemical processes in which hUev1a is involved, it is important to determine the molecular basis of the catalytic mechanism for Lys(63)-linked protein ubiquitination. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of hUev1a and its interactions with Ub and hUbc13. A structural model for the Ub-hUev1a-hUbc13-Ub tetramer was developed to gain chemical insight into the synthesis of Lys(63)-linked Ub chains. We propose that a network of hydrogen bonds involving hUbc13-Asp(81) and Ub-Glu(64) positions Ub-Lys(63) proximal to the active site. Interestingly, restrained molecular dynamics simulations in implicit solvent indicate that deprotonation of Ub-Lys(63) does not involve a general Asp or Glu base and may occur when the amino group approaches the thioester carbonyl carbon near the Bürgi-Dunitz trajectory.

Cross-Links of Quadruplex Structures from Human Telomeric DNA by Dinuclear Platinum Complexes Show the Flexibility of Both Structures

The folding of AG(3)(T(2)AG(3))(3) was investigated in the presence of Na(+) or K(+) ions, by using the dinuclear platinum complexes [{trans-PtCl(NH(3))(2)}(2)H(2)N(CH(2))(n)NH(2)]Cl(2) (n = 2 or 6). AG(3)(T(2)AG(3))(3) has been previously found to adopt two different quadruplex structures: the antiparallel one in a solution containing Na(+) and the parallel one in a K(+)-containing crystal. The two structures are strikingly distinct and are not expected to form the same platinum cross-links. Therefore, characterization of the cross-links formed with platinum complexes in solution allowed the predominant conformation(s) to be identified. The bases coordinating the platinum atoms were identified by chemical and 3'-exonuclease digestions. The observed cross-links showed that the parallel structure exists in solution whatever the cation and confirmed the existence of the antiparallel structure in the presence of both cations as previously reported from cross-linking experiments of AG(3)(T(2)AG(3))(3) by mononuclear platinum complexes. Furthermore, the major platinum cross-links were unexpectedly formed between two guanines belonging to the same G-quartet. Their formation was rationalized using molecular dynamics simulations in implicit solvent of the two quadruplex structures. It was shown that they were flexible, allowing some guanines to leave reversibly the top G-quartet and thus rendering their N(7) atom accessible to platinum complexes. Our results also suggest that the human telomere sequence could be a target for such platinum complexes.

Understanding folding and design: replica-exchange simulations of "Trp-cage" miniproteins.

Replica-exchange molecular dynamics simulations in implicit solvent have been carried out to study the folding thermodynamics of a designed 20-residue peptide, or "miniprotein." The simulations in this study used the amber (parm94) force field along with the generalized Born/solvent-accessible surface area implicit solvent model, and they spanned a range of temperatures from 273 to 630 K. Starting from a completely extended initial conformation, simulations of one peptide sequence sample conformations that are <1.0 A Calpha rms positional deviation from structures in the corresponding NMR ensemble. These folded states are thermodynamically stable with a simulated melting temperature of approximately 400 K, and they satisfy the majority of experimentally observed NMR restraints. Simulations of a related mutant peptide show a degenerate ensemble of states at low temperature, in agreement with experimental results.