Salt Bridge Formation between the I-BAR Domain and Lipids Increases Lipid Density and Membrane Curvature

Abstract

The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight "zeppelin-shaped" dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.

Figure 1

Simulated system containing I-BAR and a lipid bilayer membrane. (a) The initial arrangement of I-BAR (upper panel) and a snapshot after 200-ns MD (lower panel) in the MD1 simulation. Pink, cyan, and yellow spheres represent the headgroups of DOPC, DOPE, and DOPS, respectively. (b) Principal axes of inertia of I-BAR (three arrows). The angle θ is defined as the angle between the second principle axis of inertia (green) and the Z-axis; the latter is initially parallel to the membrane normal in MD1. (c) Initial conditions of the four MD simulations of I-BAR–membrane systems (MD1–4). ΔD _Z is defined in the main text. ‘Gap’ is the minimum heavy atom pair distance between I-BAR and the membrane along the Z-axis. The molecular graphics was created by VMD.

Figure 2

Time evolution of I-BAR binding to the lipid bilayer membrane. (a) Minimum heavy atom pair distance between the I-BAR domain and the membrane, (b) I-BAR–membrane distance ΔD _Z, (c) angle θ, and (d) curvature C _ave as a function of MD simulation time. (e,f) representative snapshots from (e) MD1 and (f) MD4. The molecular graphics was created by VMD.

Figure 3

Free energy analysis of I-BAR binding. (a) Curvature, (b) binding free energy ΔG _B, (c) free energy components ΔE _I-BAR, −TΔS _I-BAR, and ΔΔμ, (d) components of ΔE _int, (e) the number of hydrogen bonds and salt bridges within I-BAR and between I-BAR and the lipids, as a function of ΔD _Z. In (a), the broken line shows experimentally deduced curvature. (f) Thermodynamic cycle of binding/unbinding. (g) The structures of I-BAR in the crystal, in solution, and from MD1 and MD4. (h) Interaction energy between I-BAR and each lipid headgroup mapped on the XY plane. (i) Interaction energy between I-BAR with the lipid headgroups integrated along the Y-axis. (j) Lipid local density (N_lipid), (k) mean square displacement (MSD, <Δr ² (t)>) of the lipids in bilayer lateral direction at time t = 10 ns, and (l) curvature (C) as a function of the X coordinate. The horizontal bar in the bottom indicates the position of I-BAR. The results shown in (h) to (l) were obtained from 107~137 ns in MD1 and 140~170 ns in membrane only system. Center of mass for the headgroup was used to calculate lipid density, MSD, and curvature. The lipid densities and MSDs were averaged over the range of ±48.3 Å. The molecular graphics was created by VMD.

Figure 4

Effects of mutations on the membrane. (a) The number of each amino acid type and the number of each lipid type involved in binding. Open boxes indicate the total number of amino acid residues and lipids in the upper layer of the bilayer. Filled boxes represent the number of residues and lipids making contact at the I-BAR/membrane interface. (b) Positively and negatively charged residues located on the membrane binding surface. (c) Binding affinities relative to the wild-type as a function of the number of salt bridges that the wild-type can make with the lipid headgroups. The number of intra-molecular salt bridges for K108, K130, and K171 which do not make salt bridge with the lipid headgroup are also shown. (d) Intra-molecular salt bridges essential for I-BAR stability. (e) Effects of D112A, E174A and BPM mutations compared to the wild-type on HeLa cells observed by fluorescence microscopy with GFP fusion I-BAR and percentage of cells with I-BAR localization at filopodia. BPM: K142A/K143A/R145A/K146A/K147A. The molecular graphics was created by VMD.