Insight into the Mechanism of Action and Peptide-Membrane Interactions of Aib-Rich Peptides: Multitechnique Experimental and Theoretical Analysis.

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs.

Atomistic and coarse-grained simulations of membrane proteins: A practical guide.

Membrane proteins are amphipathic macromolecules whose exposed hydrophobic surfaces promote interactions with lipid membranes. Membrane proteins are remarkably diverse in terms of chemical composition and correspondingly, their biological functions and general biophysical behavior. Conventional experimental techniques provide an approach to study specific properties of membrane proteins e.g. their surface features, the nature and abundance of stabilizing intramolecular forces, preferred bilayer orientation, and the characteristics of their annular lipid shells. Molecular modeling software-and in particular, the suite of molecular dynamics algorithms-enables a more comprehensive exploration of dynamic membrane protein behavior. Molecular dynamics methods enable users to produce stepwise trajectories of proteins on arbitrary spatiotemporal scales that enable the easy identification of dynamic interactions that are beyond the scope of conventional analytical techniques. This article explains the molecular dynamics theoretical framework and popular step-by-step approaches for simulating membrane proteins in planar, and to a lesser extent, nonplanar lipid geometries. We detail popular procedures and computational tools that produce well-packed configurations of lipids and proteins and additionally, the efficient molecular dynamics simulation algorithms that reproduce their dynamic interactions.

Combining native and 'omics' mass spectrometry to identify endogenous ligands bound to membrane proteins.

Ligands bound to protein assemblies provide critical information for function, yet are often difficult to capture and define. Here we develop a top-down method, 'nativeomics', unifying 'omics' (lipidomics, proteomics, metabolomics) analysis with native mass spectrometry to identify ligands bound to membrane protein assemblies. By maintaining the link between proteins and ligands, we define the lipidome/metabolome in contact with membrane porins and a mitochondrial translocator to discover potential regulators of protein function.

To infect or not to infect: molecular determinants of bacterial outer membrane vesicle internalization by host membranes.

Outer membrane vesicles (OMVs) are spherical liposomes that are secreted by almost all forms of Gram-negative bacteria. The nanospheres contribute to bacterial pathogenesis by trafficking molecular cargo from bacterial membranes to target cells at the host-pathogen interface. We have simulated the interaction of OMVs with host cell membranes to understand why OMV uptake depends on the length of constituent lipopolysaccharide macromolecules. Using coarse-grained molecular dynamics simulations, we show that lipopolysaccharide lipid length affects OMV shape at the host-pathogen interface: OMVs with long (smooth-type) lipopolysaccharide lipids retain their spherical shape when they interact with host cell membranes, whereas OMVs with shorter (rough-type) lipopolysaccharide lipids distort and spread over the host membrane surface. In addition, we show that OMVs preferentially coordinate domain-favoring ganglioside lipids within host membranes to enhance curvature and affect the local lipid composition. We predict that these differences in shape preservation affect OMV internalization on long timescales: spherical nanoparticles tend to be completely enveloped by host membranes, whereas low sphericity nanoparticles tend to remain on the surface of cells.

Role of O-Antigen in Response to Mechanical Stress of the E. coli Outer Membrane: Insights from Coarse-Grained MD Simulations.

Lipopolysaccharide (LPS) is an important component of the outer membrane of Gram-negative bacteria, contributing to the structural integrity of the bacterial cell wall and conferring resistance to chemical attack. The rough variant of LPS contains a conserved lipid A domain and a complete core saccharide section, whereas the smooth variant additionally contains a terminal O-antigen chain. In the following, smooth LPS lipids are simulated in multicomponent membrane models using coarse-grained molecular dynamics. The simulations reveal that the lipid environment of smooth LPS lipids affects the orientation and clustering of their O-antigen chains. When the outer membrane leaflets contain smooth LPS lipids alone, the O-antigen chains are packed tightly, leading to strong cohesive intermolecular interactions. When the outer leaflets incorporate interstitial phospholipids and rough LPS variants, the O-antigen chains are tilted and less tightly bound. The different packing of terminal O-antigen chains affects lipid mobility and the mechanical strength of the Gram-negative membrane models. Gram-negative membranes with outer leaflets of smooth LPS alone can withstand surface tensions (150 mN m-1) that cause the membrane models with rough LPS lipids and comparable phospholipid bilayers to rupture much more readily.

Outer Membrane Proteins OmpA, FhuA, OmpF, EstA, BtuB, and OmpX Have Unique Lipopolysaccharide Fingerprints.

The outer membrane of Gram-negative bacteria has a highly complex asymmetrical architecture, containing a mixture of phospholipids in the inner leaflet and almost exclusively lipopolysaccharide (LPS) molecules in the outer leaflet. In E. coli, the outer membrane contains a wide range of proteins with a ß barrel architecture, that vary in size from the smallest having eight strands to larger barrels composed of 22 strands. Here we report coarse-grained molecular dynamics simulations of six proteins from the E. coli outer membrane OmpA, OmpX, BtuB, FhuA, OmpF, and EstA in a range of membrane environments, which are representative of the in vivo conditions for different strains of E. coli. We show that each protein has a unique pattern of interaction with the surrounding membrane, which is influenced by the composition of the protein, the level of LPS in the outer leaflet, and the differing mobilities of the lipids in the two leaflets of the membrane. Overall we present analyses from over 200 µs of simulation for each protein.

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 water-related 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 with MD simulations 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.

CHARMM-GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides.

A complex cell envelope, composed of a mixture of lipid types including lipopolysaccharides, protects bacteria from the external environment. Clearly, the proteins embedded within the various components of the cell envelope have an intricate relationship with their local environment. Therefore, to obtain meaningful results, molecular simulations need to mimic as far as possible this chemically heterogeneous system. However, setting up such systems for computational studies is far from trivial, and consequently the vast majority of simulations of outer membrane proteins still rely on oversimplified phospholipid membrane models. This work presents an update of CHARMM-GUI Martini Maker for coarse-grained modeling and simulation of complex bacterial membranes with lipopolysaccharides. The qualities of the outer membrane systems generated by Martini Maker are validated by simulating them in bilayer, vesicle, nanodisc, and micelle environments (with and without outer membrane proteins) using the Martini force field. We expect this new feature in Martini Maker to be a useful tool for modeling large, complicated bacterial outer membrane systems in a user-friendly manner. © 2017 Wiley Periodicals, Inc.

Through the Lipopolysaccharide Glass: A Potent Antimicrobial Peptide Induces Phase Changes in Membranes.

In the following, molecular simulations are used to reveal unexpected behavior within bacterial membranes. We show that lipopolysaccharide molecules found in these membranes form viscous amorphous solids when they are interlinked with monovalent and divalent cations. The bilayers exhibit both liquid and glassy characteristics, due to the coexistence of both liquid and crystalline domains in the bilayer. Polymyxin B1, a potent antimicrobial peptide, is shown to increase order within the lipopolysaccharide bilayers by inducing the formation of crystalline patches. Crucially we are able to decompose the energetics of insertion into their enthalpic and entropic components. The present coarse-grain molecular dynamics study provides unprecedented insights into the antibacterial action of antimicrobial peptides, thus paving the way for development of novel therapeutic agents to treat multiple drug resistant Gram-negative bacteria.

Molecular Dynamics Simulations Predict the Pathways via Which Pristine Fullerenes Penetrate Bacterial Membranes.

Carbon fullerenes are emerging as effective devices for different biomedical applications, including the transportation of nanosized drugs and extraction of harmful oxidants and radicals. It has been proposed that fullerenes could be used as novel antibacterial agents, given the realization that the nanoparticles can kill pathogenic Gram-negative bacteria. To explore this at the molecular level, we simulated C60 fullerenes with bacterial membranes using the coarse-grain molecular dynamics Martini force field. We found that pristine C60 has a limited tendency to penetrate (incomplete core) Re mutant lipopolysaccharide (LPS) leaflets, but the translocation of C60 fullerenes into (complete core) Ra mutant LPS leaflets is not thermodynamically favored. Moreover, we showed that the permeability of the Re LPS bilayers depends sensitively on the system temperature, charge of ambient ions, and prevalence of palmitoyloleoylphosphoethanolamine (POPE) defect domains. The different permeabilities are rationalized in terms of transitory head group pore formation, which underpins the translocation of C60 into the lipid core. The Re LPS lipids readily form transient micropores when they are linked with monovalent cations or when they are heated to a high temperature. POPE lipids are shown to be particularly adept at forming these transient surface cavities, and their inclusion into Re LPS membranes facilitates the formation of particularly large pores that are tunneled by C60 aggregates of a significant size (∼5 nm wide). After insertion into the lipid core, the aggregates dissociate, and the disbanded nanoparticles migrate to the interface between separate POPE and LPS domains, where they weaken the boundaries between the coexisting lipid fractions and thereby promote lipid mixing.

Structural basis for Mep2 ammonium transceptor activation by phosphorylation.

Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Šaway from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

An accurate in vitro model of the E.†coli envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.

Interaction of the antimicrobial peptide polymyxin B1 with both membranes of E. coli: a molecular dynamics study.

Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development.

An Accurate In Vitro Model of the E.†coli Envelope.

Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir-Blodgett and Langmuir-Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development.