The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

Nanoindentation response of nickel surface using molecular dynamics simulation

The mechanisms of dislocation nucleation on a nickel (Ni) (001) surface under nanoindentation behaviours are investigated using molecular dynamics simulation. The characteristic mechanisms include the molecular models of a thermal layer (TL) and thermal with a free layer (TFL), multi-step load/unload cycles, tilt angles and shapes of the indenter, and slip vectors. The model of a TL has higher reaction force than a TFL. The maximum forces of nanoindentation decrease with increasing time of the multi-step load/unload cycle. The indenter with the tilt angle has larger force to act on the molecular model than the indenter along the normal direction. The effect of the indentation shape is presented such that the conical tip has larger load force to act on the molecular model. The defects along Shockley partials on the (111) plane are produced during nanoindentation involving nucleation, glide and slip.     

Influencing the direct formation of ‘Janus’ particles in molecular dynamics simulations

The binary nucleation of phase-separated Lennard-Jones clusters was analysed under various system conditions using molecular dynamics simulations. The modified potential model provides a simple gateway to observe non-wetting behaviour and imitates the more complex interactions of non-miscible substances. Thus, not only the transition from ideally mixed clusters to so-called ‘Janus’ particles, but also the structural aspects and dynamic formation processes of nanoscopic droplets are directly observable from the gas phase. Various shapes and sizes of these inhomogeneous clusters have been found via simple tuning of system parameters. From this analysis, we gained further insight into the direct formation of ‘Janus’ particles from the gas phase.     

A molecular dynamics analysis of homogeneous bubble nucleation with a noncondensable gas in cryogenic cavitation inception

In this paper, homogeneous bubble nucleation in liquid oxygen (as one of the cryogenic fluids) with a noncondensable gas of nitrogen or that of helium was investigated using molecular dynamics method employing a fitted Lennard-Jones potential. We evaluated the influence of nitrogen gas and helium gas on the SATuration line (SAT) and the spinodal line as the thermodynamic limit of stability (TLS), and on the kinetic limit of stability (KLS) defined from a bubble nucleation rate. As a result, it was obtained that the influence of the noncondensable gases on the SAT and the TLS was negligible at molar fraction less than 1% although helium gas had several times stronger action to decrease the KLS compared with nitrogen gas. On the other hand, it was also indicated that the actual influence of both noncondensable gases on the cavitation inception in liquid oxygen might be negligible at least at standard conditions where the fluid starts to flow around or less than the atmospheric pressure.     

Stabilities and structures of islet amyloid polypeptide (IAPP22–28) oligomers: From dimer to 16-mer

Background

The formation of amyloid fibrils is associated with many age-related degenerative diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood.

Methods

Here, we performed MD simulations for the NFGAILS motif of hIAPP associated with the type II diabetes to estimate the stabilities of hIAPP_22–28 protofibrils with different sizes: from 2 to 16 chains. In addition, to study the initial self-assembly stage, 4 and 8 IAPP_22–28 chains in explicit solvent were also simulated.

Results

Our results indicate that the ordered protofibrils with no more than 16 hIAPP_22–28 chains will be structurally stable in two layers, while one-layer or three-layer models are not stable as expected. Furthermore, the oligomerization simulations show that the initial coil structures of peptides can quickly aggregate and convert to partially ordered β-sheet-rich oligomers.

Conclusions

Based on the obtained results, we found that the stability of an IAPP_22–28 oligomer was not only related with its size but also with its morphology. The driving forces to form and stabilize an oligomer are the hydrophobic effects and backbone H-bond interaction. Our simulations also indicate that IAPP_22–28 peptides tend to form an antiparallel strand orientation within the sheet.

General significance

Our finding can not only enhance the understanding about potential mechanisms of hIAPP nuclei formation and the extensive structural polymorphisms of oligomers, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.     

Atomistic modelling of interface structure and deformation mechanisms in the Al/GaN multilayer under compression

The properties of ohmic contact and thermal boundary conductance between Al and GaN have been studied extensively, but the interface structures and deformation mechanisms in the Al/GaN multilayer can be rarely found in literatures. By molecular dynamics (MD) simulations, we systematically studied the interface structures and structural deformations in the Al/GaN multilayer. Two kinds of interface structures are identified according to the different terminal surfaces of GaN; glide-set terminal interface and shuffle-set terminal interface. Further analysis shows that interface has the maximum stress and misfit lines have the maximum stress values, which serve as the dislocation sources in the Al layer due to the larger stress in the interface. The mechanical responses of the Al/GaN multilayer exhibit a minor stage and some distinctive drops in the stress–strain curve. The first stage is associated with the dislocation nucleation from the interface. Upon further compression, more slip systems appear in the Al layer and dislocation nucleation in GaN could induce drops in curves. Meanwhile, the multiplications of dislocations cause strain hardening behaviours.     

Analysis of crystal growth of methane hydrate using molecular dynamics simulation

Methane hydrate is a crystalline compound with methane molecules enclosed in cages formed by hydrogen-bonded water molecules. Understanding the mechanism of nucleation and crystal growth from methane vapour and liquid water is important for all hydrate applications. However, processes near the water/methane interface are still unclear. In this work, we focused on the crystal growth of methane hydrate seeds located near the water/methane interface. We performed molecular dynamics (MD) simulation and analysed the crystal growth of the hydrate seed at the interface. New cages formed in the liquid water phase were stabilised when they shared faces with the hydrate seed. We also investigated the crystal growth rate as the time development of the number of methane molecules trapped in hydrate cages, based on the trajectory of the MD simulation. The calculated growth rate in the direction that covers the interface was 1.38 times that in the direction towards the inside of the water phase.     

Donor-strand exchange in chaperone-assisted pilus assembly revealed in atomic detail by molecular dynamics

Adhesive multi-subunit fibres are assembled on the surface of many pathogenic bacteria via the chaperone-usher pathway. In the periplasm, a chaperone donates a β-strand to a pilus subunit to complement its incomplete immunoglobulin-like fold. At the outer membrane, this is replaced with a β-strand formed from the N-terminal extension (Nte) of an incoming pilus subunit by a donor-strand exchange (DSE) mechanism. This reaction has previously been shown to proceed via a concerted mechanism, in which the Nte interacts with the chaperone:subunit complex before the chaperone has been displaced, forming a ternary intermediate. Thereafter, the pilus and chaperone β-strands have been postulated to undergo a strand swap by a ‘zip-in-zip-out’ mechanism, whereby the chaperone strand zips out, residue by residue, as the Nte simultaneously zips in, although direct experimental evidence for a zippering mechanism is still lacking. Here, molecular dynamics simulations have been used to probe the DSE mechanism during formation of the Saf pilus from Salmonella enterica at the atomic level, allowing the direct investigation of the zip-in-zip-out hypothesis. The simulations provide an explanation of how the incoming Nte is able to dock and initiate DSE due to inherent dynamic fluctuations within the chaperone:subunit complex. In the simulations, the chaperone donor strand was seen to unbind from the pilus subunit, residue by residue, in direct support of the zip-in-zip-out hypothesis. In addition, an interaction of a residue towards the N-terminus of the Nte with a specific binding pocket (P*) on the adjacent pilus subunit was seen to stabilise the DSE product against unbinding, which also proceeded in the simulations by a zippering mechanism. Together, the study provides an in-depth picture of DSE, including the first atomistic insights into the molecular events occurring during the zip-in-zip-out mechanism.     

Evidence for proton shuffling in a thioredoxin-like protein during catalysis

Proteins of the thioredoxin (Trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. All members of the Trx family with thiol-disulfide oxidoreductase activity contain the characteristic Cys-X-X-Cys motif in their active site. Here, using Poisson-Boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigate the catalytic mechanism of DsbL, the most oxidizing Trx-like protein known to date. We observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. These results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. Intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. A consistent catalytic mechanism for DsbL, probably conferrable to other proteins of the same class, is presented. Our results suggest a functional role of hydration entropy of active-site groups.     

Stochastic Gating and Drug-Ribosome Interactions

Gentamicin is a potent antibiotic that is used in combination therapy for inhalation anthrax disease. The drug is also often used in therapy for methicillin-resistant Staphylococcusaureus. Gentamicin works by flipping a conformational switch on the ribosome, disrupting the reading head (i.e., 16S ribosomal decoding bases 1492-1493) used for decoding messenger RNA. We use explicit solvent all-atom molecular simulation to study the thermodynamics of the ribosomal decoding site and its interaction with gentamicin. The replica exchange molecular dynamics simulations used an aggregate sampling of 15 μs when summed over all replicas, allowing us to explicitly calculate the free-energy landscape, including a rigorous treatment of enthalpic and entropic effects. Here, we show that the decoding bases flip on a timescale faster than that of gentamicin binding, supporting a stochastic gating mechanism for antibiotic binding, rather than an induced-fit model where the bases only flip in the presence of a ligand. The study also allows us to explore the nonspecific binding landscape near the binding site and reveals that, rather than a two-state bound/unbound scenario, drug dissociation entails shuttling between many metastable local minima in the free-energy landscape. Special care is dedicated to validation of the obtained results, both by direct comparison to experiment and by estimation of simulation convergence.     

Computational design and experimental testing of the fastest-folding β-sheet protein

One of the most important and elusive goals of molecular biology is the formulation of a detailed, atomic-level understanding of the process of protein folding. Fast-folding proteins with low free-energy barriers have proved to be particularly productive objects of investigation in this context, but the design of fast-folding proteins was previously driven largely by experiment. Dramatic advances in the attainable length of molecular dynamics simulations have allowed us to characterize in atomic-level detail the folding mechanism of the fast-folding all-β WW domain FiP35. In the work reported here, we applied the biophysical insights gained from these studies to computationally design an even faster-folding variant of FiP35 containing only naturally occurring amino acids. The increased stability and high folding rate predicted by our simulations were subsequently validated by temperature-jump experiments. The experimentally measured folding time was 4.3 μs at 80 °C—about three times faster than the fastest previously known protein with β-sheet content and in good agreement with our prediction. These results provide a compelling demonstration of the potential utility of very long molecular dynamics simulations in redesigning proteins well beyond their evolved stability and folding speed.     

Element proportion effect on internal stress from interfaces and other microstructural components in Cu–Pb alloys

Polycrystalline materials like Cu–Pb alloy consist of four types of microstructural components, including grain cells, grain boundaries, triple junctions and vertex points, the mechanical properties of which governed by the atomic proportion of the alloy elements to a certain degree. The internal stresses from such microstructural components are quite different. Due to experimental limitations, the internal stresses from the alloy materials are difficult to measure directly, especially in the microstructural components. Here, we report a bottom-up approach using an atomistic calculation to obtain atomic properties in Cu-based alloy, as well as that in the microstructural components. The results reveal that a steep stress gradient exists at the interfaces of the alloy, which decreases significantly with the increase of the Pb. The defects evolution process in the alloy samples are investigated during tensile loading, revealing that the defect nucleation is delayed due to the decreasing von Mises stress gradient in the interfaces region as Pb increased. And the increased hydrostatic pressure in the interfaces regions, as a secondary factor can promote the defect nucleation. Among alloy samples with a grain size of 18.58 nm, that with 6.6 at.% Pb has minimal defects and the best mechanical properties.     

Heterogeneous cavitation and crystallisation with an impurity by molecular dynamics

A homogeneous metastable liquid system and a heterogeneous system with an immersed impurity were used to investigate the cavitation and crystallisation characteristics by molecular dynamics. An isochoric quench was conducted to the systems, which initially produced a cavity, where the entire system subsequently crystallised. The systems with the impurities showed an overall increase in the crystal nucleation rate and a modest but clear shape effect was observed. Similar to previous droplet condensation studies, a clear curvature effect was observed to influence the rate of the phenomenon. At the onset of quenching, crystallisation occurred on the faces of the seed, so a flat surface promoted local crystallisation, which propagated the formation of the cavity, and thereafter increased the density of the metastable liquid, so to advance overall crystallisation within the system.     

The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by 60 degrees . This recovery stroke is coupled to the activation of myosin ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a seesaw motion of the relay helix, followed by a piston-like motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery-stroke by using principal component analysis. This reveals that the only principal motions of these two helices that make a large-amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.     

On the Structure of the Proton-Binding Site in the Fo Rotor of Chloroplast ATP Synthases

The recently reported crystal structures of the membrane-embedded proton-dependent c-ring rotors of a cyanobacterial F₁F_o ATP synthase and a chloroplast F₁F_o ATP synthase have provided new insights into the mechanism of this essential enzyme. While the overall features of these c-rings are similar, a discrepancy in the structure and hydrogen-bonding interaction network of the H⁺ sites suggests two distinct binding modes, potentially reflecting a mechanistic differentiation. Importantly, the conformation of the key glutamate side chain to which the proton binds is also altered. To investigate the nature of these differences, we use molecular dynamics simulations of both c-rings embedded in a phospholipid membrane. We observe that the structure of the c₁₅ ring from Spirulina platensis is unequivocally stable within the simulation time. By contrast, the proposed structure of the H⁺ site in the chloroplast c₁₄ ring changes rapidly and consistently into that reported for the c₁₅ ring, indicating that the latter represents a common binding mode. To assess this hypothesis, we have remodeled the c₁₄ ring by molecular replacement using the published structure factors. The resulting structure provides clear evidence in support of a common binding site conformation and is also considerably improved statistically. These findings, taken together with a sequence analysis of c-subunits in the ATP synthase family, indicate that the so-called proton-locked conformation observed in the c₁₅ ring may be a common characteristic not only of light-driven systems such as chloroplasts and cyanobacteria but also of a selection of other bacterial species.     

Enzyme specificity under dynamic control II: Principal component analysis of α-lytic protease using global and local solvent boundary conditions

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190-->Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. Based on a combination of multiconformation analysis of cryo-X-ray crystallographic data, solution nuclear magnetic resonance (NMR), and normal mode calculations, we had hypothesized that the large alteration in specificity of the mutant enzyme is mainly attributable to changes in the dynamic movement of the two walls of the specificity pocket. To test this hypothesis, we performed a principal component analysis using 1-nanosecond molecular dynamics simulations using either a global or local solvent boundary condition. The results of this analysis strongly support our hypothesis and verify the results previously obtained by in vacuo normal mode analysis. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity. As shown here, concerted local movements within proteins can be efficiently analyzed through a combination of principal component analysis and molecular dynamics trajectories using a local solvent boundary condition to reduce computational time and matrix size.     

Dynamic behavior of the telomerase RNA hairpin structure and its relationship to dyskeratosis congenita

In this paper, we present the results from a comprehensive study of nanosecond-scale implicit and explicit solvent molecular dynamics simulations of the wild-type telomerase RNA hairpin. The effects of various mutations on telomerase RNA dynamics are also investigated. Overall, we found that the human telomerase hairpin is a very flexible molecule. In particular, periodically the molecule exhibits dramatic structural fluctuations represented by the opening and closing of a non-canonical base-pair region. These structural deviations correspond to significant disruptions of the direct hydrogen bonding network in the helix, widening of the major groove of the hairpin structure, and causing several U and C nucleotides to protrude into the major groove from the helix permitting them to hydrogen bond with, for example, the P3 domain of the telomerase RNA. We suggest that these structural fluctuations expose a nucleation point for pseudoknot formation. We also found that mutations in the pentaloop and non-canonical region stabilize the hairpin. Moreover, our results show that the hairpin with dyskeratosis congenita mutations is more stable and less flexible than the wild-type hairpin due to base stacking in the pentaloop. The results from our molecular dynamics simulations are in agreement with experimental observations. In addition, they suggest a possible mechanism for pseudoknot formation based on the dynamics of the hairpin structure and also may explain the mutational aspects of dyskeratosis congenita.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.