Highly Mobile Membrane Mimetic Research Articles

Improved Highly Mobile Membrane Mimetic Model for Investigating Protein-Cholesterol Interactions.

Cholesterol (CHL) plays an integral role in modulating the function and activity of various mammalian membrane proteins. Due to the slow dynamics of lipids, conventional computational studies of protein-CHL interactions rely on either long-time scale atomistic simulations or coarse-grained approximations to sample the process. A highly mobile membrane mimetic (HMMM) has been developed to enhance lipid diffusion and thus used to facilitate the investigation of lipid interactions with peripheral membrane proteins and, with customized in silico solvents to replace phospholipid tails, with integral membrane proteins. Here, we report an updated HMMM model that is able to include CHL, a nonphospholipid component of the membrane, henceforth called HMMM-CHL. To this end, we had to optimize the effect of the customized solvents on CHL behavior in the membrane. Furthermore, the new solvent is compatible with simulations using force-based switching protocols. In the HMMM-CHL, both improved CHL dynamics and accelerated lipid diffusion are integrated. To test the updated model, we have applied it to the characterization of protein-CHL interactions in two membrane protein systems, the human β2-adrenergic receptor (β2AR) and the mitochondrial voltage-dependent anion channel 1 (VDAC-1). Our HMMM-CHL simulations successfully identified CHL binding sites and captured detailed CHL interactions in excellent consistency with experimental data as well as other simulation results, indicating the utility of the improved model in applications where an enhanced sampling of protein-CHL interactions is desired.

Membrane binding and lipid-protein interaction of the C2 domain from coagulation factor V

Anchoring of coagulation factors to anionic regions of the membrane involves the C2 domain as a key player. The rate of enzymatic reactions of the coagulation factors is increased by several orders of magnitude upon membrane binding. However, the precise mechanisms behind the rate acceleration remain unclear, primarily because of a lack of understanding of the conformational dynamics of the C2-containing factors and corresponding complexes. We elucidate the membrane-bound form of the C2 domain from human coagulation factor V (FV–C2) by characterizing its membrane binding the specific lipid-protein interactions. Employing all-atom molecular dynamics simulations and leveraging the highly mobile membrane-mimetic (HMMM) model, we observed spontaneous binding of FV-C2 to a phosphatidylserine (PS)-containing membrane within 2–25 ns across twelve independent simulations. FV-C2 interacted with the membrane through three loops (spikes 1–3), achieving a converged, stable orientation. Multiple HMMM trajectories of the spontaneous membrane binding provided extensive sampling and ample data to examine the membrane-induced effects on the conformational dynamics of C2 as well as specific lipid-protein interactions. Despite existing crystal structures representing presumed “open” and “closed” states of FV-C2, our results revealed a continuous distribution of structures between these states, with the most populated structures differing from both “open” and “closed” states observed in crystal environments. Lastly, we characterized a putative PS-specific binding site formed by K23, Q48, and S78 located in the groove enclosed by spikes 1–3 (PS-specificity pocket), suggesting a different orientation of a bound headgroup moiety compared to previous proposals based upon analysis of static crystal structures.

Efficient sampling of TrkA transmembrane domain dimerization captures functionally distinct structural ensembles.

Upon ligand binding, the transmembrane domains (TMD) of the receptor tyrosine kinase (RTK) dimerize, induce conformational change in the juxtamembrane domain (JMD), and trigger downstream phosphorylation that eventually determines fate of the cell. To overcome the experimental difficulty in probing the TMD dimerization events in membrane bilayers, we employed all-atom molecular dynamics with a novel membrane mimetic solvent Simple Carbon Solvent Ethane (SCSE) to efficiently sample the TMD dimerization processes for tropomyosin receptor kinase A (TrkA). Spontaneous and stable TMD dimers were observed in less than 50 ns of simulation time from an initially separated grid configuration. We then performed hierarchical clustering using a customized distance metric based on TMD contact residues, and revealed major structural ensembles of TMD dimers. We demonstrated the validity of the resolved TMD dimer structures by performing MM-GBSA free energy calculation and molecular dynamics with full-tail lipid bilayers. As the residues in contact in the most populated TMD dimer ensemble from our SCSE simulations overlap with those found to be critical for kinase activation, we hypothesized that the captured TMD dimer corresponds to the active state, while the previously characterized NMR structure is the inactive state. To understand the role of different TMD dimer conformations in downstream signaling, we assembled TMD+JMD constructs using the captured in simulation dimer structure and the NMR structure. We modelled the system with Highly Mobile Membrane Mimetic (HMMM) for accelerated peptide-lipid interactions and found that the captured TMD dimer leads to JMD conformations distinct from those tethered to the NMR dimer structure, which may enable the kinase domain for cross-phosphorylation. Our method represents a new approach to study RTK dimerization and sample physiologically relevant dimer structures.

Lactaderhin's C1 domain restricts protein's ensemble as it binds to charged membranes.

Lactadherin's (Lact) binding to phosphotidylserine (PS) enriched cell surfaces is critical for controlling our blood coagulation. Although structural and mutagenesis studies indicate important binding regions of this protein, they are not sufficient to construct a detailed, membrane-bound model especially since they mainly consider only one of its two globular domains, namely C2. We thus use molecular dynamics (MD) simulations and the AlphaFold program to generate an ensemble of Lact encounter complexes with a negatively charged bilayer consistent with biochemical studies. This was accomplished by first using the highly mobile membrane mimetic (HMMM) to efficiently simulate its C2 domain binding to PS lipids. We then converted the HMMM model to standard full-tail lipid membranes to further sample and obtain converged bound structures that we call the “vertical” and “side-lying” ensembles. From these bilayer-bound poses, we infer which one allows for attachment of its other C1 domain and use AlphaFold to reconstruct it and further simulate to capture equilibrium complexes. Modeling revealed that C1 domain restricts the dynamics structural ensemble of Lact bound to membranes. We also report Lact's full dynamic structure in the presence of negatively charged lipids and compare them with other homologous blood coagulation proteins to provide a rationale for its competitive binding. These results shed light on the structural mechanism of blood coagulation by considering its binding in a membrane environment which can serve as a general framework for studying these critical protein-membrane interactions. They also provide a structural basis for further understanding the significance that PS lipids play in regulating blood coagulation processes.

Modeling the membrane binding mechanism of a lipid transport protein Osh4 to single membranes

All-atom (AA) molecular dynamics simulations are used to unravel the binding mechanism of yeast oxysterol binding protein (Osh4) to model membranes with varying anionic lipid concentration using AA and the highly mobile membrane mimetic (HMMM) representations. For certain protein-lipid interactions, an improved forcefield description is used (CUFIX) to accurately describe lipid-protein electrostatic interactions. Our detailed computational studies have identified a single, β-crease oriented, membrane-bound conformation of Osh4 for all anionic membranes. The penetration of the PHE-239 residue below the membrane phosphate plane is the characteristic signature of the membrane-bound state of Osh4. As the phenylalanine loop anchors itself deeply in the membrane; the other regions of the Osh4, namely, ALPS motif, β6- β7 loop, β14- β15 loop, and β16- β17 loop, maximize their contact with the membrane. Furthermore, loose lipid packing and higher mobility of HMMM enable stronger association of the ALPS motif with the membrane lipids through its hydrophobic surface. After the HMMM is converted to AA and equilibrated, the binding is two to three times stronger compared with simulations started with the AA representation, yielding the major importance of the ALPS motif to binding. Quantitative estimation of binding energy revealed that the phenylalanine loop plays a crucial role in stable membrane attachment of Osh4 and contributes significantly toward overall binding process. The CUFIX parameters provide a more balanced picture of hydrophobic and electrostatic interactions between the protein and the membrane, which differs from our past work that showed salt bridges alone stabilized Osh4-membrane contact. Our study provides a comprehensive picture of the binding mechanism of Osh4 with model single membranes and, thus, understanding of the initial interactions is important for elucidating the biological function of this protein to shuttle lipids between organelles.

Extrinsic Complex Structure Developed through Novel Simulation Methodology: Key Putative Atomic-Level Interactions Identified

The ternary extrinsic complex of the coagulation pathway (Tissue Factor (TF): Factor VIIa (FVIIa): Factor X (FX)) is critically important to the regulation of blood clotting. Activation of FX in this membrane-bound complex is vital to both the major pathways which produce the explosive burst of thrombin required for clot formation to proceed. Despite high interest in this complex as a potential drug target for hemophilia and thrombosis, little has been determined experimentally concerning its structure due to the lipid-dependent nature of its formation. As the complex will only form on the surface of a membrane with a specific composition of anionic phospholipids, experimental structure determination through NMR or X-ray crystallography is prohibitively difficult.We will present the first atomic-level resolution structure of the ternary complex to include membrane interactions, developed using a novel methodology which combines protein-protein docking and extensive non-equilibrium molecular dynamics simulations. Over 560,000 protein-only structures predicting interactions between key domains of the extrinsic complex were first developed using the DOT2 protein docking program. Top docked structures were selected based on agreement with experimental information, and used to identify residues of FX and TF:FVIIa likely to interact. Protein-protein contact information from docking was then used to developed biased non-equilibrium simulations in which the complex was induced to form along a particular pathway. Using five non-equilibrium simulations of 200 ns, binding between membrane-bound structures of FX and TF:FVIIa was slowly induced. The highly mobile membrane mimetic (HMMM) model, an advanced membrane model which replaces slow-moving lipid tails with detergent molecules, was used to allow increased sampling of protein-lipid interacts during this deliberate complex formation. Each of the five resulting complexes was simulated for an additional 200 ns without biases, and putative key protein-protein and protein-lipid interactions involved in complex formation were identified. Residues on the membrane-binding domain of FX where found in the simulations to bind residues of TF previously identified experimentally as critical for complex formation. This structure provides not only the best available information on protein binding for the extrinsic complex but the only atomic-level information on the protein-lipid interactions which are crucial for its formation. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Beta-2-Glycoprotein I Domain V interaction with Plasma Membrane

Beta-2-Glycoprotein I (β2GPI), also known as apolipoprotein H, is a 48kDa protein which circulates in blood at a high concentration [0.2 mg/ml]. β2GPI is the main target of autoantibodies in antiphospholipid syndrome (APS) which results in thrombosis and/or recurrent miscarriage. Therefore, investigating membrane binding mechanism of β2GPI is important to elucidate how this protein become immunogenic upon binding to plasma membrane and to help target specific drug development for APS. It is known that β2GPI binds to anionic phospholipids in the plasma membrane via its positively charged domain V (DV) but the mechanism of binding is still elusive. in this study, we performed multiple 500-ns molecular dynamics simulations of β2GPI-DV in the presence of membranes containing PC and PS in varying compositions employing the HMMM (highly mobile membrane mimetic) model. We observed stable membrane binding in eight out of ten replicas. Our results show that in seven out of eight replicas, β2GPI-DV interacts with the membrane via its flexible loop (311-317) which insert into the membrane below the PO4 level. interestingly, in one replica we observe insertion of the loop carrying three lysine residues, a mode which closely corresponds to an alternative binding mode reported previously. The orientation of membrane-bound states was characterized by measuring the tilt angle between the protein and the membrane. After insertion of the flexible loop, β2GPI-DV held an average tilt angle of 92 with respect to the membrane normal. This study establishes the significance of the flexible loop for membrane binding of β2GPI which could lead to a more rational approach in drug design for treatment of APS.

Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular Membranes

Infection of human cells by the SARS-CoV2 relies on its binding to a specific receptor and subsequent fusion of the viral and the host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an extensive array of molecular dynamics (MD) simulations taking advantage of the Highly Mobile Membrane Mimetic (HMMM) model, to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing human cellular membranes at an atomic level, and to characterize its membrane-bound form.

Microscopic Characterization of GRP1 PH Domain Interaction with Anionic Membranes.

The pleckstrin homology (PH) domain of general receptor for phosphoionositides 1 (GRP1-PHD) binds specifically to phosphatidylinositol (3,4,5)-triphosphate (PIP3 ), and acts as a second messenger. Using an extensive array of molecular dynamics (MD) simulations employing highly mobile membrane mimetic (HMMM) model as well as complementary full membrane simulations, we capture differentiable binding and dynamics of GRP1-PHD in the presence of membranes containing PC, PS, and PIP3 lipids in varying compositions. While GRP1-PHD forms only transient interactions with pure PC membranes, incorporation of anionic lipids resulted in stable membrane-bound configurations. We report the first observation of two distinct PIP3 binding modes on GRP1-PHD, involving PIP3 interactions at a "canonical" and at an "alternate" site, suggesting the possibility of simultaneous binding of multiple anionic lipids. The full membrane simulations confirmed the stability of the membrane bound pose of GRP1-PHD as captured from our HMMM membrane binding simulations. By performing additional steered membrane unbinding simulations and calculating nonequilibrium work associated with the process, as well as metadynamics simulations, on the protein bound to full membranes, allowing for more quantitative examination of the binding strength of the GRP1-PHD to the membrane, we demonstrate that along with the bound PIP3 , surrounding anionic PS lipids increase the energetic cost of unbinding of GRP1-PHD from the canonical mode, causing them to dissociate more slowly than the alternate mode. Our results demonstrate that concurrent binding of multiple anionic lipids by GRP1-PHD contributes to its membrane affinity, which in turn control its signaling activity. © 2019 Wiley Periodicals, Inc.

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain.

In the receptor tyrosine kinase family, conformational change induced by ligand binding is transmitted across the membrane via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with highly mobile membrane mimetic (HMMM) to capture the native conformation of the JMD in tropomyosin receptor kinase A (TrkA). We find that phosphatidylinositol 4,5-bisphosphate (PIP2) lipids engage in stable binding with multiple basic residues. Anionic lipids can compete with salt bridges within the peptide and alter TrkA-JMD conformation. We discover three-residue insertion into the membrane and are able to either enhance or reduce the level of insertion through computationally-designed point mutations. The vesicle-binding experiment supports computational results and indicates that hydrophobic insertion is comparable to electrostatic binding for membrane anchoring. Biochemical assays on cell lines with mutated TrkA show that enhanced TrkA-JMD insertion promotes receptor degradation but does not affect the short-term signaling capacity. Our joint work points to a scenario where lipid headgroups and tails interact with basic and hydrophobic residues on disordered domain, respectively, to restrain flexibility and potentially modulate protein function.

Anionic Lipid-Dependent Gliding of Cytochrome C across Bioenergetic Membranes

Bioenergetic membranes found in mitochondria, thylakoids, and chromatophores are primary sites of ATP production in living cells. These membranes contain an electron transport chain (ETC) in which electrons are shuttled between a series of redox proteins during the generation of ATP via oxidative phosphorylation. Classical examples of electron shuttles include members of the cytochrome c family. Phospholipid composition plays a role in determining the local electrostatic environment of the surface of the owing to the spatial distribution of their charged head groups. Cardiolipin (CDL) is a characteristic phospholipid in bioenergetic membranes, and is a significant contributor of negative surface charge. Interactions between cytochromes and phospholipid head groups in the membrane can in principle affect the rate of travel between ETC components, influencing the rate of ATP turnover. We used Highly Mobile Membrane Mimetic (HMMM) simulations of cytochrome c2 (cyt.c2) to study protein-lipid interactions in the context of diffusion across a model bioenergetic membrane (PC:PE:PG:CDL = 10:30:46:14). Using HMMM, we extend the time-scales of molecular dynamics to aggressively sample protein and lipid diffusion and interactions at an atomic resolution. We observed a bias for cyt.c2 to bind anionic lipids, with a clear preference for CDL. During diffusion, cyt.c2 maintains a relatively fixed tilt with respect to the membrane normal with wider fluctuations in the angle with respect to axes in the plane of the membrane, caused by its prominent dipole moment. The cyt.c2 diffusion coefficient calculated from simulation trajectories is 0.7 Å2 /ns. Our simulation supports a mode of diffusion in which cyt.c2 skips across the membrane, rather than a purely two dimensional, on the surface of the membrane, or a three dimensional mode of diffusion.

Effect of Membrane Lipid Packing on Stable Binding of the ALPS Peptide.

The amphipathic lipid packing sensor (ALPS) motif, originally discovered on the ArfGAP1 membrane-binding protein, binds to pre-existing large packing defects in a membrane (spontaneous or due to membrane curvature), though a more precise relationship between the ALPS peptide and packing defect characteristics of a membrane remains unclear. We developed an image processing technique for identifying packing defects to quantify the relationship between the ALPS peptide of the Osh4 protein in yeast and packing defects on a membrane model using molecular dynamics simulations. We used the highly mobile membrane mimetic (HMMM) model to create very large packing defects and expedite the binding time scale. Most prominently, we show that the probability of the ALPS peptide moving toward the membrane increases when it is near a large packing defect. Deviations from this trend exist for very large packing defects (≳115 Å2), which we propose is due to an overwhelming hydrophobic effect and a reduced electrostatic effect when large portions of the nonpolar core are exposed and the peptide is oriented unfavorably. Furthermore, we compared our HMMM results to similar simulations using all-atom lipid membranes. The binding time scales of the ALPS peptide can be reduced by roughly 1 order of magnitude when HMMM is used, while still maintaining many of the important physical characteristics of the binding process observed when using an all-atom lipid membrane.

Setting Up All-Atom Molecular Dynamics Simulations to Study the Interactions of Peripheral Membrane Proteins with Model Lipid Bilayers.

All-atom molecular dynamics (MD) simulations enable the study of biological systems at atomic detail, complement the understanding gained from experiment, and can also motivate experimental techniques to further examine a given biological process. This method is based on statistical mechanics; it predicts the trajectory of atoms over time by solving Newton's Laws of motion taking into account all forces. Here, we describe the use of this methodology to study the interaction between peripheral membrane proteins and a lipid bilayer. Specifically, we provide step-by-step instructions to set up MD simulations to study the binding and interaction of the amphipathic helix of Osh4, a lipid transport protein, and Thanatin, an antimicrobial peptide (AMP), with model lipid bilayers using both fully detailed lipid tails and the highly mobile membrane-mimetic (HMMM) method to enhance conformational sampling.

Identification of Cardiolipin Binding Sites on Membrane Proteins using an Accelerated Computational Membrane Model

Cardiolipin (CDL), a negatively-charged lipid found primarily in bacterial and mitochondrial membranes, is believed to play a major role in membrane morphology, some hereditary heart diseases, as well as acting as a proton reservoir in the inner mitochondrial membrane. CDL has been shown to regulate many integral membrane proteins involved in bioenergetics, often binding with nanomolar affinity. In this study we incorporate CDL into the Highly Mobile Membrane Mimetic (HMMM), a membrane model consisting of lipid head groups partitioned by an organic solvent mimicking hydrophobic acyl tails, which allows for fast lipid diffusion, nanosecond spontaneous membrane insertion of proteins, and providing atomistic sampling of CDL-protein interactions, which gives rise to the identification of potential CDL binding residues. As an application, we chose CLC chloride transporter (CLC-ec1), an integral membrane protein involved in maintaining proton gradients, which initial experimental results show that CDL increases CLC-ec1 activity, likely due to CDLs ability to act as a proton reservoir. The increased lipid diffusion and sampling of CDL containing HMMM membranes aided in determining potential binding sites for ClC Chloride transporter. Incorporating CDL into the HMMM model involved applying harmonic restraints to each carbonyl carbon for each tail to maintain membrane thickness, as well as applying RMSD based collective variable restraints on ClC-ec1. Using ten one-hundred nanosecond HMMM simulations of CLC-ec1 embedded into a 80:20 POPE:CDL membrane, interactions between CDL and protein residues were measured. The overall microsecond simulation time, together with the enhanced dynamics offered by the HMMM membrane, provided a large sample size, which led to narrowing down the potential CDL binding sites to two sites, each stabilized by two basic side chains, thus expediting and guiding ClC-ec1 mutagenesis studies.

Investigating Cholesterol Dynamics and Interactions with the Dopamine Transporter using a Membrane Mimetic Model

Cholesterol, an integral component of animal lipid membranes, is involved in modulation of both membrane physical properties and a wide variety of membrane proteins. Conventional computational studies of cholesterol-protein interactions have been either using coarse-grained methods or relying on > μs all-atom simulations due to the slow dynamics of lipids, in order to provide useful information. The highly mobile membrane-mimetic (HMMM) model has been previously reported to study interactions between peripheral membrane proteins and phospholipids with expedited lipid reorganization. Here, we have extended the use of the HMMM model to investigate interactions between integral membrane protein and cholesterol. Cholesterol dynamics were first closely examined in the HMMM model with extensive all-atom molecular dynamics simulations. Both lateral diffusion and flip-flop motion were shown to be significantly accelerated at cholesterol concentrations ranging from 5% to 50%, while atomic density profiles and internal dynamics of cholesterols with proper restraints in the HMMM membranes were found similar to those in full-length lipid membranes. The HMMM model was then applied to study cholesterol interactions with an integral membrane protein, dopamine transporter. The cholesterol binding sites and detailed interactions with the dopamine transporter observed in the HMMM membrane were compared with those from microsecond all-atom molecular dynamics simulations and experimental data. Our results show great potential and advantages of using the HMMM model to identify cholesterol binding sites with enhanced efficiency and clarify cholesterol-protein interactions without losing atomic details.

Molecular Simulations Provide Insite on Lysine Snorkeling Modulation of the Integrin Transmembrane Domain

Integrins are signaling proteins involved in many critical cellular functions, including cell adhesion and motility. Their activation process is triggered by the dissociation of α and β transmembrane domains. Integrin αIIbβ3 is involved in aggregation of platelets during blood clotting. Mutagenesis of a single lysine, K716, on the β3 transmembrane domain has been shown to lead to spontaneous integrin αIIbβ3 activation, although the mechanism of this activation has yet to be fully elucidated. Atomic-level molecular dynamics simulations have been performed on wild-type β3 as well as K716E and K716A mutants inserted into a phospholipid bilayer, allowing for detailed characterization of the lipid-protein interactions though which K716 modulation of αIIbβ3 activation occurs. Use of a specialized membrane model, the highly mobile membrane mimetic (HMMM), in which the membrane core is replaced with solvent molecules and the lipid head groups are retained at atomic resolution, has allowed for enhanced sampling of critical protein-lipid interactions. Fifteen total simulations of 50 ns each have been performed, five for each of the wild type and mutant structures. Each HMMM simulation was then converted to full membrane and simulated for an additional 50 ns. The wild type β3 domains converged to a larger angle to the membrane than did the mutant structures, a finding consistent with the results of experimental mutagenesis studies. It was found that lysine snorkeling allows for critical interactions which control tilt of the domain in the membrane. This study has allowed for detailed characterization of the protein-lipid interactions involved in modulating β3 tilt to the membrane and, ultimately, αIIbβ3 activation.

Anionic Lipids Take Charge: Juxtamembrane Domain Interactions with Cellular Membrane

Nearly half of the human integral membrane proteome consists of single-pass transmembrane domains. Of these, receptor tyrosine kinases (RTKs), a class with twenty families of cell-surface receptors, are key regulators of vital cellular processes and targets of drug development efforts. The fibroblast growth factor receptors (FGFRs) a family of RTKs that influences cell growth, proliferation, differentiation, is also activated in a number of cancers. FGFRs dynamic architecture, that contains extracellular, single-pass transmembrane (TM), juxtamembrane (JM), and kinase domains, is tightly intertwined with cellular membrane and is a challenging target for structural studies. Recent experimental studies revealed that highly charged FGFR3 JM domains are involved in stabilization of unliganded FGFR3 dimers, which is achieved in part through interactions with charged lipids of the inner leaflet of the cellular membrane. The mechanism of JM-lipid interaction is unknown. We use all-atom molecular dynamics with highly mobile membrane mimetic (HMMM) model to capture spontaneous JM-lipid encounter complexes. To investigate the role of the anionic lipids we performed extensive simulations of multiple replicas of pure phosphatidylcholine (PC), binary mixtures of phosphatidylserine (PS) and PC, and tertiary mixtures of PC, PS, and phosphatidylinositol 4,5-bisphosphate (PIP2) lipid bilayers. Simulations revealed formation of stable anionic lipid conformations coordinated by charged JM residues. Lipid type-specific geometries lead to characteristic modes of JM-lipid interactions revealing the role of the charged lipid headgroups in establishing specific interactions. Knowledge of these dynamic structures will facilitate our understanding of mechanisms of RTK activation.

Lipid specificity of the membrane binding domain of coagulation factor X

Background Factor X (FX) binds to cell membranes in a highly phospholipid-dependent manner and, in complex with tissue factor and factor VIIa (FVIIa), initiates the clotting cascade. Experimental information concerning the membrane-bound structure of FX with atomic resolution has remained elusive because of the fluid nature of cellular membranes. FX is known to bind preferentially to phosphatidylserine (PS). Objectives To develop the first membrane-bound model of the FX-GLA domain to PS at atomic level, and to identify PS-specific binding sites of the FX-GLA domain. Methods Molecular dynamics (MD) simulations were performed to develop an atomic-level model for the FX-GLA domain bound to PS bilayers. We utilized a membrane representation with enhanced lipid mobility, termed the highly mobile membrane mimetic (HMMM), permitting spontaneous membrane binding and insertion by FX-GLA in multiple 100-ns simulations. In 14 independent simulations, FX-GLA bound spontaneously to the membrane. The resulting membrane-bound models were converted from HMMM to conventional membrane and simulated for an additional 100ns. Results The final membrane-bound FX-GLA model allowed for detailed characterization of the orientation, insertion depth and lipid interactions of thedomain, providing insight into the molecular basis of its PS specificity. All binding simulations converged to thesame configuration despite differing initial orientations. Conclusions Analysis of interactions between residues in FX-GLA and lipid-charged groups allowed for potential PS-specific binding sites to be identified. This new structural and dynamic information provides an additional step towards a full understanding of the role of atomic-level lipid-protein interactions in regulating the critical and complex clotting cascade.

Spontaneous membrane insertion of a dengue virus NS2A peptide

Non-structural NS2A protein of Dengue virus is essential for viral replication but poorly characterized because of its high hydrophobicity. We have previously shown experimentally that NS2A possess a segment, peptide dens25, known to insert into membranes and interact specifically with negatively-charged phospholipids. To characterize its membrane interaction we have used two types of molecular dynamics membrane model systems, a highly mobile membrane mimetic (HMMM) and an endoplasmic reticulum (ER) membrane-like model. Using the HMMM system, we have been able of demonstrating the spontaneous binding of dens25 to the negatively-charged phospholipid 1,2-divaleryl-sn-glycero-3-phosphate containing membrane whereas no binding was observed for the membrane containing the zwitterionic one 1,2-divaleryl-sn-glycero-3-phosphocholine. Using the ER-like membrane model system, we demonstrate the spontaneous insertion of dens25 into the middle of the membrane, it maintained its three-dimensional structure and presented a nearly parallel orientation with respect to the membrane surface. Both charged and hydrophobic amino acids, presenting an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment, are responsible for membrane binding and insertion. Dens25 might control protein/membrane interaction and be involved in membrane rearrangements critical for the viral cycle. These data should help us in the development of inhibitor molecules that target NS2A segments involved in membrane reorganisation.

Extension of the Highly Mobile Membrane Mimetic to Transmembrane Systems through Customized in Silico Solvents.

The mechanics of the protein-lipid interactions of transmembrane proteins are difficult to capture with conventional atomic molecular dynamics, due to the slow lateral diffusion of lipids restricting sampling to states near the initial membrane configuration. The highly mobile membrane mimetic (HMMM) model accelerates lipid dynamics by modeling the acyl tails nearest to the membrane center as a fluid organic solvent while maintaining an atomic description of the lipid headgroups and short acyl tails. The HMMM has been applied to many peripheral protein systems; however, the organic solvent used to date caused deformations in transmembrane proteins by intercalating into the protein and disrupting interactions between individual side chains. We ameliorate the effect of the solvent on transmembrane protein structure through the development of two new in silico Lennard-Jones solvents. The parameters for the new solvents were determined through an extensive parameter search in order to match the bulk properties of alkanes in a highly simplified model. Using these new solvents, we substantially improve the insertion free energy profiles of 10 protein side chain analogues across the entire bilayer. In addition, we reduce the intercalation of solvent into transmembrane systems, resulting in native-like transmembrane protein structures from five different topological classes within a HMMM bilayer. The parametrization of the solvents, in addition to their computed physical properties, is discussed. By combining high lipid lateral diffusion with intact transmembrane proteins, we foresee the developed solvents being useful to efficiently identify membrane composition inhomogeneities and lipid binding caused by the presence of membrane proteins.