Thomas J. Piggot

Molecular Dynamics Simulations of Phosphatidylcholine Membranes: A Comparative Force Field Study

Molecular dynamics simulations provide a route to studying the dynamics of lipid bilayers at atomistic or near atomistic resolution. Over the past 10 years or so, molecular dynamics simulations have become an established part of the biophysicists tool kit for the study of model biological membranes. As simulation time scales move from tens to hundreds of nanoseconds and beyond, it is timely to re-evaluate the accuracy of simulation models. We describe a comparative analysis of five freely available force fields that are commonly used to model lipid bilayers. We focus our analysis on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. We show that some bilayer properties have a pronounced force field dependence, while others are less sensitive. In general, we find strengths and weaknesses, with respect to experimental data, in all of the force fields we have studied. We do, however, find some combinations of simulation and force field parameters that should be avoided when simulating DPPC and POPC membranes. We anticipate that the results presented for some of the membrane properties will guide future improvements for several force fields studied in this work.

Electroporation of the E. coli and S. Aureus membranes: molecular dynamics simulations of complex bacterial membranes.

Bacterial membranes are complex organelles composed of a variety of lipid types. The differences in their composition are a key factor in determining their relative permeabilities. The success of antibacterial agents depends upon their interaction with bacterial membranes, yet little is known about the molecular-level interactions within membranes of different bacterial species. To address this, we have performed molecular dynamics simulations of two bacterial membranes: the outer membrane of E. coli and the cell membrane of S. aureus . We have retained the chemical complexity of the membranes by considering the details of their lipidic components. We identify the extended network of lipid-lipid interactions that stabilize the membranes. Our simulations of electroporation show that the S. aureus cell membrane is less resistant to poration than the E. coli outer membrane. The mechanisms of poration for the two membranes have subtle differences; for the E. coli outer membrane, relative differences in mobilities of the lipids of both leaflets are key in the process of poration.

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.

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor

Background: Toll-like receptor 4 (TLR4) in complex with MD-2 stimulates innate immunological pathways in response to bacterial lipopolysaccharide (LPS). Results: Molecular simulations reveal the mechanism of TLR4 complex signaling in response to agonists or antagonists. Conclusion: Conserved clamshell motions in MD-2 allosterically signal ligand-bound state via the conserved phenylalanine 126 residue to TLR4. Significance: The structural basis for molecular switching during endotoxin-induced TLR4 activation is revealed in atomic detail. As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling.

Conformational dynamics and membrane interactions of the E. coli outer membrane protein FecA: A molecular dynamics simulation study

The TonB-dependent transporters mediate high-affinity binding and active transport of a variety of substrates across the outer membrane of Escherichia coli. The substrates transported by these proteins are large, scarce nutrients that are unable to gain entry into the cell by passive diffusion across the complex, asymmetric bilayer that constitutes the outer membrane. Experimental studies have identified loop regions that are essential for the correct functioning of these proteins. A number of these loops have been implicated in ligand binding. We report the first simulations of an E. coli outer membrane protein in an asymmetric model membrane that incorporates lipopolysaccharide (LPS) molecules. Comparative simulations of the apo and holo forms of the TonB-dependent transporter FecA in different membrane models enable us to identify the nature of the LPS-protein interactions and determine how these interactions impact upon the conformational dynamics of this protein. In particular, our simulations provide molecular-level insights into the influence of the environment and ligand on the dynamics of the functionally important loops of FecA. In addition, we provide insights into the nature of the protein-ligand interactions and ligand induced conformational change in FecA.

Stability and membrane orientation of the fukutin transmembrane domain: a combined multiscale molecular dynamics and circular dichroism study.

The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids.

The NorM MATE Transporter from N. gonorrhoeae: Insights into Drug and Ion Binding from Atomistic Molecular Dynamics Simulations

The multidrug and toxic compound extrusion transporters extrude a wide variety of substrates out of both mammalian and bacterial cells via the electrochemical gradient of protons and cations across the membrane. The substrates transported by these proteins include toxic metabolites and antimicrobial drugs. These proteins contribute to multidrug resistance in both mammalian and bacterial cells and are therefore extremely important from a biomedical perspective. Although specific residues of the protein are known to be responsible for the extrusion of solutes, mechanistic details and indeed structures of all the conformational states remain elusive. Here, we report the first, to our knowledge, simulation study of the recently resolved x-ray structure of the multidrug and toxic compound extrusion transporter, NorM from Neisseria gonorrhoeae (NorM_NG). Multiple, atomistic simulations of the unbound and bound forms of NorM in a phospholipid lipid bilayer allow us to identify the nature of the drug-protein/ion-protein interactions, and secondly determine how these interactions contribute to the conformational rearrangements of the protein. In particular, we identify the molecular rearrangements that occur to enable the Na(+) ion to enter the cation-binding cavity even in the presence of a bound drug molecule. These include side chain flipping of a key residue, GLU-261 from pointing toward the central cavity to pointing toward the cation binding side when bound to a Na(+) ion. Our simulations also provide support for cation binding in the drug-bound and apo states of NorM_NG.

Stability and membrane interactions of an autotransport protein: MD simulations of the Hia translocator domain in a complex membrane environment

Hia is a trimeric autotransporter found in the outer membrane of Haemphilus influenzae. The X-ray structure of Hia translocator domain revealed each monomer to consist of an α-helix connected via a loop to a 4-stranded β-sheet, thus the topology of the trimeric translocator domain is a 12-stranded β-barrel containing 3 α-helices that protrude from the mouth of the β-barrel into the extracellular medium. Molecular dynamics simulations of the Hia monomer and trimer have been employed to explore the interactions between the helices, β-barrel and connecting loops that may contribute to the stability of the trimer. In simulations of the Hia monomer we show that the central α-helix may stabilise the fold of the 4-stranded β-sheet. In simulations of the Hia trimer, a H-bond network involving residues in the β-barrel, α-helices and loops has been identified as providing stability for the trimeric arrangement of the monomers. Glutamine residues located in the loops connecting the α-helices to the β-barrel are orientated in a triangular arrangement such that each forms 2 hydrogen bonds to each of the corresponding glutamines in the other loops. In the absence of the loops, the β-barrel becomes distorted. Simulations show that while the trimeric translocator domain β-barrel is inherently flexible, it is unlikely to accommodate the passenger domain in a folded conformation. Simulations of Hia in an asymmetric model of the outer membrane have revealed membrane-protein interactions that anchor the protein within its native membrane environment.

Probing the oligomeric state and interaction surfaces of Fukutin-I in dilauroylphosphatidylcholine bilayers

Fukutin-I is localised to the endoplasmic reticulum or Golgi apparatus within the cell, where it is believed to function as a glycosyltransferase. Its localisation within the cell is thought to to be mediated by the interaction of its N-terminal transmembrane domain with the lipid bilayers surrounding these compartments, each of which possesses a distinctive lipid composition. However, it remains unclear at the molecular level how the interaction between the transmembrane domains of this protein and the surrounding lipid bilayer drives its retention within these compartments. In this work, we employed chemical cross-linking and fluorescence resonance energy transfer measurements in conjunction with multiscale molecular dynamics simulations to determine the oligomeric state of the protein within dilauroylphosphatidylcholine bilayers to identify interactions between the transmembrane domains and to ascertain any role these interactions may play in protein localisation. Our studies reveal that the N-terminal transmembrane domain of Fukutin-I exists as dimer within dilauroylphosphatidylcholine bilayers and that this interaction is driven by interactions between a characteristic TXXSS motif. Furthermore residues close to the N-terminus that have previously been shown to play a key role in the clustering of lipids are shown to also play a major role in anchoring the protein in the membrane.

Dynamics of Crowded Vesicles: Local and Global Responses to Membrane Composition.

The bacterial cell envelope is composed of a mixture of different lipids and proteins, making it an inherently complex organelle. The interactions between integral membrane proteins and lipids are crucial for their respective spatial localization within bacterial cells. We have employed microsecond timescale coarse-grained molecular dynamics simulations of vesicles of varying sizes and with a range of protein and lipid compositions, and used novel approaches to measure both local and global system dynamics, the latter based on spherical harmonics analysis. Our results suggest that both hydrophobic mismatch, enhanced by embedded membrane proteins, and curvature based sorting, due to different modes of undulation, may drive assembly in vesicular systems. Interestingly, the modes of undulation of the vesicles were found to be altered by the specific protein and lipid composition of the vesicle. Strikingly, lipid dynamics were shown to be coupled to proteins up to 6 nm from their surface, a substantially larger distance than has previously been observed, resulting in multi-layered annular rings enriched with particular types of phospholipid. Such large protein-lipid complexes may provide a mechanism for long-range communication. Given the complexity of bacterial membranes, our results suggest that subtle changes in lipid composition may have major implications for lipid and protein sorting under a curvature-based membrane-sorting model.