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Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.

The Escherichia coli UraA H+-uracil symporter is a member of the nucleobase/ascorbate transporter (NAT) family of proteins, and is responsible for the proton-driven uptake of uracil. Multiscale molecular dynamics simulations of the UraA symporter in phospholipid bilayers consisting of: 1) 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC); 2) 1-palmitoyl 2-oleoyl-phosphatidylethanolamine (POPE); and 3) a mixture of 75% POPE, 20% 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG); and 5% 1-palmitoyl 2-oleoyl-diphosphatidylglycerol/cardiolipin (CL) to mimic the lipid composition of the bacterial inner membrane, were performed using the MARTINI coarse-grained force field to self-assemble lipids around the crystal structure of this membrane transport protein, followed by atomistic simulations. The overall fold of the protein in lipid bilayers remained similar to the crystal structure in detergent on the timescale of our simulations. Simulations were performed in the absence of uracil, and resulted in a closed state of the transporter, due to relative movement of the gate and core domains. Anionic lipids, including POPG and especially CL, were found to associate with UraA, involving interactions between specific basic residues in loop regions and phosphate oxygens of the CL head group. In particular, three CL binding sites were identified on UraA: two in the inner leaflet and a single site in the outer leaflet. Mutation of basic residues in the binding sites resulted in the loss of CL binding in the simulations. CL may play a role as a "proton trap" that channels protons to and from this transporter within CL-enriched areas of the inner bacterial membrane.

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.

Membrane proteins are frequently present in crowded environments, which favour lateral association and, on occasions, two-dimensional crystallization. To better understand the non-specific lateral association of a membrane protein we have characterized the free energy landscape for the dimerization of a bacterial outer membrane protein, NanC, in a phospholipid bilayer membrane. NanC is a member of the KdgM-family of bacterial outer membrane proteins and is responsible for sialic acid transport in E. coli. Umbrella sampling and coarse-grained molecular dynamics were employed to calculate the potentials of mean force (PMF) for a variety of restrained relative orientations of two NanC proteins as the separation of their centres of mass was varied. We found the free energy of dimerization for NanC to be in the range of -66 kJ mol(-1) to -45 kJ mol(-1). Differences in the depths of the PMFs for the various orientations are related to the shape of the proteins. This was quantified by calculating the lipid-inaccessible buried surface area of the proteins in the region around the minimum of each PMF. The depth of the potential well of the PMF was shown to depend approximately linearly on the buried surface area. We were able to resolve local minima in the restrained PMFs that would not be revealed using conventional umbrella sampling. In particular, these features reflected the local organization of the intervening lipids between the two interacting proteins. Through a comparison with the distribution of lipids around a single freely-diffusing NanC, we were able to predict the location of these restrained local minima for the orientational configuration in which they were most pronounced. Our ability to make this prediction highlights the important role that lipid organization plays in the association of two NanCs in a bilayer.

Integrins are heterodimeric (αβ) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the α/β integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin α/β TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin α and β transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.

Coarse-grained molecular dynamics simulations of the E. coli outer membrane proteins FhuA, LamB, NanC, OmpA and OmpF in a POPE/POPG (3:1) bilayer were performed to characterise the diffusive nature of each component of the membrane. At small observation times (<10 ns) particle vibrations dominate phospholipid diffusion elevating the calculated values from the longer time-scale bulk value (>50 ns) of 8.5×10(-7) cm(2) s(-1). The phospholipid diffusion around each protein was found to vary based on distance from protein. An asymmetry in the diffusion of annular lipids in the inner and outer leaflets was observed and correlated with an asymmetry in charged residues in the vicinity of the inner and outer leaflet head-groups. Protein rotational and translational diffusion were also found to vary with observation time and were inversely correlated with the radius of gyration of the protein in the plane of the bilayer. As the concentration of protein within the bilayer was increased, the overall mobility of the membrane decreased reflected in reduced lipid diffusion coefficients for both lipid and protein components. The increase in protein concentration also resulted in a decrease in the anomalous diffusion exponent α of the lipid. Formation of extended clusters and networks of proteins led to compartmentalisation of lipids in extreme cases.

The association of hemagglutinin (HA) with lipid rafts in the plasma membrane is an important feature of the assembly process of influenza virus A. Lipid rafts are thought to be small, fluctuating patches of membrane enriched in saturated phospholipids, sphingolipids, cholesterol and certain types of protein. However, raft-associating transmembrane (TM) proteins generally partition into Ld domains in model membranes, which are enriched in unsaturated lipids and depleted in saturated lipids and cholesterol. The reason for this apparent disparity in behavior is unclear, but model membranes differ from the plasma membrane in a number of ways. In particular, the higher protein concentration in the plasma membrane may influence the partitioning of membrane proteins for rafts. To investigate the effect of high local protein concentration, we have conducted coarse-grained molecular dynamics (CG MD) simulations of HA clusters in domain-forming bilayers. During the simulations, we observed a continuous increase in the proportion of raft-type lipids (saturated phospholipids and cholesterol) within the area of membrane spanned by the protein cluster. Lateral diffusion of unsaturated lipids was significantly attenuated within the cluster, while saturated lipids were relatively unaffected. On this basis, we suggest a possible explanation for the change in lipid distribution, namely that steric crowding by the slow-diffusing proteins increases the chemical potential for unsaturated lipids within the cluster region. We therefore suggest that a local aggregation of HA can be sufficient to drive association of the protein with raft-type lipids. This may also represent a general mechanism for the targeting of TM proteins to rafts in the plasma membrane, which is of functional importance in a wide range of cellular processes.

Bacterial chemoreceptors provide an important model for understanding signalling processes. In the serine receptor Tsr from E. coli, a binding event in the periplasmic domain of the receptor dimer causes a shift in a single transmembrane helix of roughly 0.15 nm towards the cytoplasm. This small change is propagated through the ≈ 22 nm length of the receptor, causing downstream inhibition of the kinase CheA. This requires interactions within a trimer of receptor dimers. Additionally, the signal is amplified across a 53,000 nm(2) array of chemoreceptor proteins, including ≈ 5,200 receptor trimers-of-dimers, at the cell pole. Despite a wealth of experimental data on the system, including high resolution structures of individual domains and extensive mutagenesis data, it remains uncertain how information is communicated across the receptor from the binding event to the downstream effectors. We present a molecular model of the entire Tsr dimer, and examine its behaviour using coarse-grained molecular dynamics and elastic network modelling. We observe a large bending in dimer models between the linker domain HAMP and coiled-coil domains, which is supported by experimental data. Models of the trimer of dimers, built from the dimer models, are more constrained and likely represent the signalling state. Simulations of the models in a 70 nm diameter vesicle with a biologically realistic lipid mixture reveal specific lipid interactions and oligomerisation of the trimer of dimers. The results indicate a mechanism whereby small motions of a single helix can be amplified through HAMP domain packing, to initiate large changes in the whole receptor structure.

Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃). The interaction of GRP1-PH with PI(3,4,5)P₃ in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P₃-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5)P₃ molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P₃ within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P₃, forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors.

The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

ABC transporters are a large family of membrane proteins involved in a variety of cellular processes, including multidrug and tumor resistance and ion channel regulation. Advances in the structural and functional understanding of ABC transporters have revealed that hydrolysis at the two canonical nucleotide-binding sites (NBSs) is co-operative and non-simultaneous. A conserved core architecture of bacterial and eukaryotic ABC exporters has been established, as exemplified by the crystal structure of the homodimeric multidrug exporter Sav1866. Currently, it is unclear how sequential ATP hydrolysis arises in a symmetric homodimeric transporter, since it implies at least transient asymmetry at the NBSs. We show by molecular dynamics simulation that the initially symmetric structure of Sav1866 readily undergoes asymmetric transitions at its NBSs in a pre-hydrolytic nucleotide configuration. MgATP-binding residues and a network of charged residues at the dimer interface are shown to form a sequence of putative molecular switches that allow ATP hydrolysis only at one NBS. We extend our findings to eukaryotic ABC exporters which often consist of two non-identical half-transporters, frequently with degeneracy substitutions at one of their two NBSs. Interestingly, many residues involved in asymmetric conformational switching in Sav1866 are substituted in degenerate eukaryotic NBS. This finding strengthens recent suggestions that the interplay of a consensus and a degenerate NBS in eukaroytic ABC proteins pre-determines the sequence of hydrolysis at the two NBSs.