Finding a needle in a haystack: the role of electrostatics in target lipid recognition by PH domains

Abstract

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.

Figure 1. Structure of the GRP1 PH domain.

A The crystal structure of GRP1-PH (PDB 1FGY). The I(1,3,4,5)P₄ headgroup and the sidechains of two key residues (K279 and R284) are shown as van der Waals spheres. The protein is oriented such that the bilayer normal, as determined by previous MD simulations, is vertical (i.e. defines the z axis). B Electrostatic potential of GRP1-PH projected onto the solvent-accessible surface, showing the large positive electrostatic potential around the I(1,3,4,5)P₄ binding site. The electrostatic potential was calculated in the absence of I(1,3,4,5)P₄ using APBS as described in the main text and is coloured from −5 kT/e (red) to +5 kT/e (blue). The protein is shown in the same orientation as in A and the molecular dipole moment (calculated using the Protein Dipole Moments Server (http://bioinfo.weizmann.ac.il/dipol/; [70]) is indicated by a black arrow. C Snapshot from a BD simulation showing the protein solvent-accessible surface coloured by electrostatic potential, with the molecular dipole moment shown as a black arrow as in B. The POPC lipid bilayer is shown as a white surface with the single PI(3,4,5)P₃ molecule shown in black.

Figure 2. Schematic diagram of the BD simulation setup.

The b-sphere is truncated to form an open hemispherical surface situated on one side of the membrane, with the value of q set such that all trajectories are terminated before the protein is able to diffuse outside the perimeter of the membrane patch. The three coordinates r, z and θ specify the position and orientation of the protein relative to the PI(3,4,5)P₃ headgroup.

Figure 3. Lipid bilayer models.

A Electrostatic potential isocontours of a POPC lipid bilayer (red = negative, blue = positive), with a central PI(3,4,5)P₃ molecule clearly visible as a region of negative electrostatic potential (indicated by a white circle). B Electrostatic potential isocontours after modifying lipids with an evenly distributed fractional charge. Each lipid headgroup of the upper (i.e. cytoplasmic) leaflet of the bilayer has a fractional charge of −0.4 e. C Isocontours after a randomly selected subset of 40% of the lipids in the upper leaflet were assigned a charge of −1.0 e.

Figure 4. Positional steering of GRP1-PH for the initial bilayer model.

A Distribution of radial locations, r, of GRP1-PH over the course of the 5000 BD simulations in each ensemble for the case of evenly distributed, fractional charges ranging from 0.0 e (no anionic lipids) to 1.0 e (all anionic lipids in the cytoplasmic leaflet of the bilayer). The centre of mass of the ‘target’ PI(3,4,5)P₃ headgroup is located at r = 0. The inset graph shows the half width at half maximum (HWHM) for each of the r distributions, calculated by fitting a single Gaussian centred at r = 0 to each data set. The HWHM is plotted against the fractional negative charge assigned to the lipid headgroups. B Distribution of d positions of the protein for the same set of even charge distribution BD simulations. The centre of mass of the PI(3,4,5)P₃ headgroup, defining the surface of the bilayer, lies at d = 0.

Figure 5. Positional steering of GRP1-PH using alternative lipid configurations.

A Distribution of radial locations, r, for the BD simulations based on randomly distributed, integer-valued negative charges on the lipid headgroups. The centre of mass of the ‘target’ PI(3,4,5)P₃ headgroup is located at r = 0. B Distribution of radial locations, r, for the BD simulations based on the lipid configurations generated from a CGMD simulation of a lipid bilayer containing 20% anionic lipids.

Figure 6. Comparing positional steering for different lipid configurations.

Comparison of the profile generated by averaging the five r distributions (Figure 5B) from the BD simulations using the lipid configurations produced from a CGMD simulation with a 20% anionic lipid concentration (purple) with the r profile from the BD simulations using evenly distributed fractional charges of −0.2 e across all lipids (red).

Figure 7. Orientational steering of GRP1-PH.

A Distribution of orientations, θ, of GRP1-PH over the course of the BD simulations for the case of the evenly spread, fractional charges from 0.0 to −1.0 e. The dotted line at θ = 56° corresponds to the angle, θ, that the molecular dipole moment makes with respect to the z axis in the membrane bound complex. B Two-dimensional distributions of θ vs. r from the BD simulations with the fractional charge distributions with all lipids (other than PI(3,4,5)P₃) given a charge of −0.2 e. Note that the origin corresponds to the configuration obtaining by manual docking plus simulations (see main text and [28]). C Distribution of radial ‘first encounter’ locations for the BD simulations with evenly spread, fractional charges from 0.0 to −1.0 e.

Figure 8. MD simulations of bound complex formation.

A An optimal configuration for subsequent membrane-binding extracted from the ensemble of BD simulations conducted using an uncharged, zwitterionic lipid bilayer containing PI(3,4,5)P₃. In this case r = 9 Å, d = 18 Å and θ = 27°. B A snapshot at the end of a subsequent 100 ns MD simulation. C, D Fingerprint plots showing the location of PH domain residues contacting the phosphorus atoms of PI(3,4,5)P₃ in C the X-ray structure (PDB 1FGY) and D the 100 ns snapshot from the MD simulation shown in B. In each plot the minimum distances between each residue and each phosphorus atom are shown.