Bridging the N-terminal and middle domains in FliG of the flagellar rotor

Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function

Molecular dynamics simulations of bacterial outer membrane lipid extraction: Adequate sampling?

The outer membrane of Gram-negative bacteria is almost exclusively composed of lipopolysaccharide in its outer leaflet, whereas the inner leaflet contains a mixture of phospholipids. Lipopolysaccharide diffuses at least an order of magnitude slower than phospholipids, which can cause issues for molecular dynamics simulations in terms of adequate sampling. Here, we test a number of simulation protocols for their ability to achieve convergence with reasonable computational effort using the MARTINI coarse-grained force-field. This is tested in the context both of potential of mean force (PMF) calculations for lipid extraction from membranes and of lateral mixing within the membrane phase. We find that decoupling the cations that cross-link the lipopolysaccharide headgroups from the extracted lipid during PMF calculations is the best approach to achieve convergence comparable to that for phospholipid extraction. We also show that lateral lipopolysaccharide mixing/sorting is very slow and not readily addressable even with Hamiltonian replica exchange. We discuss why more sorting may be unrealistic for the short (microseconds) timescales we simulate and provide an outlook for future studies of lipopolysaccharide-containing membranes. </p

A pH-dependent cluster of charges in a conserved cryptic pocket on flaviviral envelopes

Flaviviruses are enveloped viruses which include human pathogens that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making vaccine-based routes of fighting infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it has the potential to diminish the effects of ADE. We examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of raft systems that represent a substantial portion of the flaviviral envelope. We utilised a benzene-mapping approach that led to a discovery of shared hotspots and conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited strain-specific characteristics. An alternative conserved cryptic site at the E protein domain interfaces showed a consistent dynamic behaviour across flaviviruses and contained a conserved cluster of ionisable residues. Constant-pH simulations revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer

Single-molecule studies of flavivirus envelope dynamics: Experiment and computation

10.1016/j.pbiomolbio.2018.09.001PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY14338-5

Motional clustering in supra-τc conformational exchange influences NOE cross-relaxation rate

Biomolecular spin relaxation processes, such as the NOE, are commonly modeled by rotational τc-tumbling combined with fast motions on the sub-τc timescale. Motions on the supra-τc timescale, in contrast, are considered to be completely decorrelated to the molecular tumbling and therefore invisible. Here, we show how supra-τc dynamics can nonetheless influence the NOE build-up between methyl groups. This effect arises because supra-τc motions can cluster the fast-motion ensembles into discrete states, affecting distance averaging as well as the fast-motion order parameter and hence the cross-relaxation rate. We present a computational approach to estimate methyl–methyl cross-relaxation rates from extensive (&gt;100 7τc) all-atom molecular dynamics (MD) trajectories on the example of the 723-residue protein Malate Synthase G. The approach uses Markov state models (MSMs) to resolve transitions between metastable states and thus to discriminate between sub-τc and supra-τc conformational exchange. We find that supra-τc exchange typically increases NOESY cross-peak intensities. The methods described in this work extend the theory of modeling sub-μs dynamics in spin relaxation and thus contribute to a quantitative estimation of NOE cross-relaxation rates from MD simulations, eventually leading to increased precision in structural and functional studies of large proteins

The disordered plant dehydrin Lti30 protects the membrane during water-related stress by cross-linking lipids

10.1074/jbc.RA118.007163JOURNAL OF BIOLOGICAL CHEMISTRY294166468-648

A Benzene-Mapping Approach for Uncovering Cryptic Pockets in Membrane-Bound Proteins

Molecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM force field using the GROMACS simulation package to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This method, for which we have made implementation files freely available, relies on modified nonbonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in the solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-$-d-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell

The disordered plant dehydrin Lti30 protects the membrane during water-related stress by cross-linking lipids

Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and waterrelated stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane- binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together withMDsimulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.</p