Self-assembling of peptide/membrane complexes by atomistic molecular dynamics simulations

Abstract

Model biological membranes consisting of peptide/lipid-bilayer complexes can nowadays be studied by classical molecular dynamics (MD) simulations at atomic detail. In most cases, the simulation starts with an assumed state of a peptide in a preformed bilayer, from which equilibrium configurations are difficult to obtain due to a relatively slow molecular diffusion. As an alternative, we propose an extension of reported work on the self-organization of unordered lipids into bilayers, consisting of including a peptide molecule in the initial random configuration to obtain a membrane-bound peptide simultaneous to the formation of the lipid bilayer. This strategy takes advantage of the fast reorganization of lipids, among themselves and around the peptide, in an aqueous environment. Model peptides of different hydrophobicity, CH3-CO-W2L18W2-NH2 (WL22) and CH3-CO-W2A18W2-NH2 (WA22), in dipalmitoyl-phosphatidylcholine (DPPC) are used as test cases. In the equilibrium states of the peptide/membrane complexes, achieved in time ranges of 50-100 ns, the two peptides behave as expected from experimental and theoretical studies. The strongly hydrophobic WL22 is inserted in a transmembrane configuration and the marginally apolar, alanine-based WA22 is found in two alternative states: transmembrane inserted or parallel to the membrane plane, embedded close to the bilayer interface, with similar stability. This shows that the spontaneous assembly of peptides and lipids is an unbiased and reliable strategy to produce and study models of equilibrated peptide/lipid complexes of unknown membrane-binding mode and topology.

FIGURE 1

Snapshots of the spontaneous aggregation of a mixture of DPPC lipids, water, and the WL22 peptide (simulation 1 of Table 1). Headgroup atoms are depicted blue, atoms of the lipid tails are depicted light gray, and water molecules are drawn in green. The backbone of the peptide is shown in a simplified tube representation with dark gray color. The side chains of Trp residues are in red. For clarity, the figures do not exactly correspond to actual simulation boxes but show part of the system repeated in space. The initial random distribution of molecules (a) evolves into micelle-like clusters that concentrate in a distinct area, where water starts to be excluded ((b), 1.5 ns). Extensive fusion and ordering yields a metastable bilayer structure where the two leaflets are fused at the level of a transbilayer lipid pore, here shown at time 35 ns (c). The pore eventually closes (at 37 ns), and the membrane is further equilibrated up to 50 ns (d). During the aggregation process, the peptide accompanies the lipids through hydrophobic interactions, first as part of a big, micelle-like cluster (b). As the primordial bilayer forms, the peptide reorients (c) and keeps inserted across the membrane for the rest of the simulation (d).

FIGURE 2

Density distribution of characteristic groups during the self-assembling of peptide/bilayer complexes, resolved along the direction normal to the membrane formed at the end of the process. Boxes a–d and e–h correspond to simulations 1 (complex with the WL22 peptide) and 7 (complex with the WA22 peptide), respectively. The solid line marks densities of lipid headgroup atoms, dotted lines are for lipid acyl tails, dashed lines for water molecules, and dotted-dashed lines for peptide atoms. Four different averages are shown, with labels corresponding to the same stages as in Fig. 1: (a and e), time interval 0–200 ps; (b and f), time 1–2 ns; (c and g), time 30–40 ns; and (d and h), final equilibrated complexes (time 45–50 ns and 135–140 ns, respectively). The positional order characteristic of a lipid bilayer can be seen in c, d, g, and h, although in the first two cases the water density across the membrane indicates the presence of a pore. The density of peptide atoms in the final stages shows clearly its position across the membrane (WL22, (d)) or bound parallel to the membrane reaching both the hydrocarbon and interface regions (WA22, (h)).

FIGURE 3

Water accessible surface for hydrophobic groups in lipid (a) and WL22 peptide (b) molecules in simulation 1. (a) Lipid acyl tail accessibility: the exposed carbon tail surface is quickly lowered within the first nanosecond (inset, logarithmic timescale) and subsequent reduction attenuates. A final small step toward lower accessibility occurs after ∼15 ns, due to the ordering of lipids into a liquid crystal bilayer. (b) Hydrophobic peptide accessibility: From the beginning, the peptide is closely surrounded by nonordered lipids, which largely excludes water and keeps accessible area low and fairly constant during the first ∼30 ns. After this time, accessibility decreases to a lower rung, coinciding with a fluctuation of the peptide orientation (Fig. 6 b) just before the pore closes (37 ns).

FIGURE 4

Evolution of the number of clusters throughout the self-assembly process for simulations 10 (no peptide, (a)) and 1 (with peptide WL22, (b and c)). The inset in b corresponds to the number of clusters of simulation 1 but calculated considering only lipids. Two lipids (or a peptide and a lipid) are defined to be in the same cluster if the distance between their center of mass is smaller than 1.1 nm. (a and b) The number of clusters decreases at an irregular pace. At the end of the process two clusters are formed, corresponding to the two monolayers (a and inset in b), which reduce to one cluster if the peptide is considered in the peptide/membrane complex (b). (c) Size of the cluster including the peptide. The peptide cluster size does not increase significantly until the first nanosecond, when small and irregular lipid-only clusters coalesce massively into it.

FIGURE 5

Formation of the liquid-crystal bilayer seen as the time evolution of an order parameter of lipid acyl tails (S_l, see Methods). The increase of S_l proceeds in a step-wise manner, with fast and slow phases overlapping with other important events of self-assembly (see text).

FIGURE 6

Tilt angle of lipid chains () and peptide molecules () during self-assembly. (a) Distribution of of sn1 and sn2 lipid acyl-chains in a 10-ns time window of the equilibrated WL22/membrane complex corresponding to simulation 1. Time evolution of (black) and the mean value of the distribution of (gray) for simulations 1 (b) and 7 (c).