Ion-Induced PIP2 Clustering with Martini3: Modification of Phosphate-Ion Interactions and Comparison with CHARMM36.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a critical lipid for cellular signaling. The specific phosphorylation of the inositol ring controls protein binding as well as clustering behavior. Two popular models to describe ion-mediated clustering of PIP2 are Martini3 (M3) and CHARMM36 (C36). Molecular dynamics simulations of PIP2-containing bilayers in solutions of potassium chloride, sodium chloride, and calcium chloride, and at two different resolutions are performed to understand the aggregation and the model parameters that drive it. The average M3 clusters of PIP2 in bilayers of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and PIP2 bilayers in the presence of K+, Na+, or Ca2+ contained 2.2, 2.6, and 6.4 times more PIP2 than C36 clusters, respectively. Indeed, the Ca2+-containing systems often formed a single large aggregate. Reparametrization of the M3 ion-phosphate Lennard-Jones interaction energies to reproduce experimental osmotic pressure of sodium dimethyl phosphate (DMP), K[DMP], and Ca[DMP]2 solutions, the same experimental target as C36, yielded comparably sized PIP2 clusters for the two models. Furthermore, C36 and the modified M3 predict similar saturation of the phosphate groups with increasing Ca2+, although the coarse-grained model does not capture the cooperativity between K+ and Ca2+. This characterization of the M3 behavior in the presence of monovalent and divalent ions lays a foundation to study cation/protein/PIP2 clustering.

Short hydrocarbon stapled ApoC2-mimetic peptides activate lipoprotein lipase and lower plasma triglycerides in mice.

Introduction: Defects in lipolysis can lead to hypertriglyceridemia, which can trigger acute pancreatitis and is also associated with cardiovascular disease. Decreasing plasma triglycerides (TGs) by activating lipoprotein lipase (LPL) with ApoC2 mimetic peptides is a new treatment strategy for hypertriglyceridemia. We recently described a dual ApoC2 mimetic/ApoC3 antagonist peptide called D6PV that effectively lowered TG in several mouse models but has limitations in terms of drug development. The aim of this study was to create the next generation of ApoC2 mimetic peptides. Methods: We employed hydrocarbon staples, as well as select amino acid substitutions, to make short single helical mimetic peptides based on the last helix of ApoC2. Peptides were first tested for their ability to activate LPL and then in hypertriglyceridemia mouse models. All-atom simulations of peptides were performed in a lipid-trilayer model of TG-rich lipoproteins to discern their possible mechanism of action. Results: We designed a single stapled peptide called SP1 (21 residues), and a double stapled (stitched) peptide called SP2 (21 residues) and its N-terminal acylated analogue, SP2a. The hydrocarbon staples increased the amphipathicity of the peptides and their ability to bind lipids without interfering with LPL activation. Indeed, from all-atom simulations, the conformations of SP1 and SP2a are restrained by the staples and maintains the proper orientation of the LPL activation motif, while still allowing their deeper insertion into the lipid-trilayer model. Intraperitoneal injection of stapled peptides (1-5 umoles/kg) into ApoC2-hypomorphic mice or human ApoC3-transgenic resulted in an 80%-90% reduction in plasma TG within 3 h, similar to the much longer D6PV peptide (41 residues). Other modifications (replacement L-Glu20, L-Glu21 with their D-isomers, N-methylation of Gly19, Met2NorLeu and Ala1alpha-methylAla substitutions, N-terminal octanoylation) were introduced into the SP2a peptide. These changes made SP2a highly resistant to proteolysis against trypsin, pepsin, and Proteinase K, while maintaining similar efficacy in lowering plasma TG in mice. Conclusion: We describe a new generation of ApoC2 mimetic peptides based on hydron carbon stapling that are at least equally potent to earlier peptides but are much shorter and resistant to proteolysis and could be further developed into a new therapy for hypertriglyceridemia.

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions.

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

Finite-Size Effects in Simulations of Peptide/Lipid Assembly.

Molecular dynamics simulations are an attractive tool for understanding lipid/peptide self-assembly but can be plagued by inaccuracies when the system sizes are too small. The general guidance from self-assembly simulations of homogeneous micelles is that the total number of surfactants should be three to five times greater than the equilibrium aggregate number of surfactants per micelle. Herein, the heuristic is tested on the more complicated self-assembly of lipids and amphipathic peptides using the Cooke and Martini 3 coarse-grained models. Cooke model simulations with 50 to 1000 lipids and no peptide are dominated by finite-size effects, with usually one aggregate (micelle or nanodisc) containing most of the lipids forming at each system size. Approximately 200 systems of different peptide/lipid (P/L) ratios and sizes of up to 1000 lipids yield a "finite-size phase diagram" for peptide driven self-assembly, including a coexistence region of micelles and discs. Insights from the Cooke model are applied to the assembly of dimyristoylphosphatidylcholine and the ELK-neutral peptide using the Martini 3 model. Systems of 150, 450, and 900 lipids with P/L = 1/6.25 form mixtures of lipid-rich discs that agree in size with experiment and peptide-rich micelles. Only the 150-lipid system shows finite-size effects, which arise from the long-tailed distribution of aggregate sizes. The general rule of three to five times the equilibrium aggregate size remains a practical heuristic for the Cooke and Martini 3 systems investigated here.

Coarse-Grained Force Fields from the Perspective of Statistical Mechanics: Better Understanding of the Origins of a MARTINI Hangover.

The popular MARTINI coarse-grained model is used as a test case to analyze the adherence of top-down coarse-grained molecular dynamics models (i.e., models primarily parametrized to match experimental results) to the known features of statistical mechanics for the underlying all-atom representations. Specifically, the temperature dependence of various pair distribution functions, and hence their underlying potentials of mean force via the reversible work theorem, are compared between MARTINI 2.0, Dry MARTINI, and all-atom simulations mapped onto equivalent coarse-grained sites for certain lipid bilayers. It is found that the MARTINI models do not completely capture the lipid structure seen in atomistic simulations as projected onto the coarse-grained mappings and that issues of accuracy and temperature transferability arise due to an incorrect enthalpy-entropy decomposition of these potentials of mean force. The potential of mean force for the association of two amphipathic helices in a lipid bilayer is also calculated, and especially at shorter ranges, the MARTINI and all-atom projection results differ substantially. The former is much less repulsive and hence will lead to a higher probability of MARTINI helix association in the MARTINI bilayer than occurs in the actual all-atom case. Additionally, the bilayer height fluctuation spectra are calculated for the MARTINI model, and compared to the all-atom results, it is found that the magnitude of thermally averaged amplitudes at intermediate length scales are quite different, pointing to a number of possible consequences for realistic modeling of membrane processes. Taken as a whole, the results presented here show disagreement in the enthalpic and entropic driving forces driving lateral structure in lipid bilayers as well as quantitative differences in association of embedded amphipathic helices, which can help direct future efforts to parametrize CG models with better agreement to the all-atom systems they aspire to represent.

Lipid-Composition-Mediated Forces Can Stabilize Tubular Assemblies of I-BAR Proteins.

Collective action by inverse-Bin/Amphiphysin/Rvs (I-BAR) domains drive micron-scale membrane remodeling. The macroscopic curvature sensing and generation behavior of I-BAR domains is well characterized, and computational models have suggested various mechanisms on simplified membrane systems, but there remain missing connections between the complex environment of the cell and the models proposed thus far. Here, we show a connection between the role of protein curvature and lipid clustering in the relaxation of large membrane deformations. When we include phosphatidylinositol 4,5-bisphosphate-like lipids that preferentially interact with the charged ends of an I-BAR domain, we find clustering of phosphatidylinositol 4,5-bisphosphate-like lipids that induce a directional membrane-mediated interaction between membrane-bound I-BAR domains. Lipid clusters mediate I-BAR domain interactions and cause I-BAR domain aggregates that would not arise through membrane fluctuation-based or curvature-based interactions. Inside of membrane protrusions, lipid cluster-mediated interaction draws long side-by-side aggregates together, resulting in more cylindrical protrusions as opposed to bulbous, irregularly shaped protrusions.

Unusual Organization of I-BAR Proteins on Tubular and Vesicular Membranes.

Protein-mediated membrane remodeling is a ubiquitous and critical process for proper cellular function. Inverse Bin/Amphiphysin/Rvs (I-BAR) domains drive local membrane deformation as a precursor to large-scale membrane remodeling. We employ a multiscale approach to provide the molecular mechanism of unusual I-BAR domain-driven membrane remodeling at a low protein surface concentration with near-atomistic detail. We generate a bottom-up coarse-grained model that demonstrates similar membrane-bound I-BAR domain aggregation behavior as our recent Mesoscopic Membrane with Explicit Proteins model. Together, these models bridge several length scales and reveal an aggregation behavior of I-BAR domains. We find that at low surface coverage (i.e., low bound protein density), I-BAR domains form transient, tip-to-tip strings on periodic flat membrane sheets. Inside of lipid bilayer tubules, we find linear aggregates parallel to the axis of the tubule. Finally, we find that I-BAR domains form tip-to-tip aggregates around the edges of membrane domes. These results are supported by in vitro experiments showing low curvature bulges surrounded by I-BAR domains on giant unilamellar vesicles. Overall, our models reveal new I-BAR domain aggregation behavior in membrane tubules and on the surface of vesicles at low surface concentration that add insight into how I-BAR domain proteins may contribute to certain aspects of membrane remodeling in cells.

Ionic Liquids Can Selectively Change the Conformational Free-Energy Landscape of Sugar Rings.

We investigated the conformational free energy landscape of glucose solvated in water and in the ionic liquids (ILs) 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) and 1-butyl-3-methylimidazoulim boron tetrafluoride ([Bmim][BF4]). To quantify equilibrium thermodynamic solvent effects, molecular dynamics simulations in conjunction with enhanced sampling based on the metadynamics framework were used. The results show that the solvent choice induces significant differences in the equilibrium ring structures, which may help further resolve the molecular mechanism governing IL-mediated cellulose dissolution.