Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes

Abstract

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP₃-containing membranes. The free energy of interaction of the PH domain with more than two PIP₃ molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.

Fig. 1. MD simulations for calculating PMFs of PH/PIP₃ interactions.

(A) Structure of the GRP1 PH domain (Protein Data Bank ID: 1FGY; yellow cartoon format) with an Ins(1,3,4,5)P₄ molecule (ball and stick format) bound at the canonical site (CA). The approximate location of the noncanonical site (NCA) is indicated. (B) Collective variable (CV) based on the minimum distance between the protein and lipid as used in the REUS-MD simulations. (C) Potentials of mean force (PMFs) for the GRP1 PH domain interacting with lipid bilayers containing from 1 to 10 PIP₃ molecules, showing the free energy of interaction as a function of protein-lipid minimum distance. The three vertical dashed lines correspond to the protein-lipid minimum distances of 0.48, 0.73, and 1.5 nm (see text for details).

Fig. 2. Free energy surfaces of the GRP1 PH domain interacting with a lipid bilayer including 3 PIP₃ molecules in each leaflet.

E, B_A, and B_B refer to the Encounter, Bound_A and Bound_B states of the PH domain when interacting with the membrane (see main text and Fig. 5B for further details). Three different projections of the free energy landscape are shown: (A) as a function of cosθ (where θ is the angle between a vector corresponding to the PH domain α helix and the z axis perpendicular to the membrane) and the protein-membrane COM distance, (B) as a function of cosθ and the protein-membrane minimum distance, and (C) as a function of the protein-membrane COM distance and the corresponding minimum distance.

Fig. 3. Free energy surfaces of the GRP1 PH domain interacting with a lipid bilayer including 1 to 10 PIP₃ molecules in each leaflet.

The free energy landscapes are shown as a function of cosθ and the protein-membrane COM distance.

Fig. 4. Clustering of lipids in the bilayer (xy) plane underneath a bound GRP1 PH domain.

The density (unbiased density normalized by the maximum density) of phosphate headgroups of PIP₃ molecules in the bilayer plane corresponding to each bound state is shown on a heat map scale from dark red to yellow. Peaks in the density corresponding to interactions with the canonical binding site (CA), the noncanonical binding site (NCA), and a third site (3rd; see main text for details) are shown. Density maps are shown for the PH domain in the Bound_B and Bound_A configurations interacting with a lipid bilayer including 3 PIP₃ molecules in each leaflet and in the Bound_A configuration interacting with a lipid bilayer including 5 PIP₃ molecules in each leaflet (in this latter case, the protein is not shown in the interests of clarity). Bottom: Probability density functions (PDFs) for different numbers of PIP₃ molecules bound to the PH domain in each of the states.

Fig. 5. A mechanism of PH binding proceeding via an encounter state to two bound states.

(A) Depth of minimum in the PMF (see also Fig. 1C) of the GRP1 PH domain as a function of the number of PIP₃ molecules in each leaflet of the bilayer. Data points for the WT PH domain are shown for simulations using REUS and 1 to 10 PIP₃ molecules in each leaflet of the bilayer (blue). The red points correspond to REUS simulations of the K273A mutant. (B) Schematic of a three-step mechanism for binding of the GRP1 PH domain to a bilayer containing multiple PIP₃ molecules (see main text for details).