Free Energies of Interaction of pH Domains with Phosphatidylinositol Phosphate Lipids

Abstract

Interactions of peripheral membrane proteins with membranes, often mediated by lipid-binding modules such as Pleckstrin Homology (PH) domains, are vital for many signal transduction and related processes in cells. PH domains have been shown to associate with phosphatidylinositol phosphate (PIP) lipids, binding to different PIP species with differing affinities and selectivity. However, the exact molecular and energetic details of the PH-membrane interactions remain in many cases elusive. Potential of mean force calculations, using an umbrella sampling approach, allow quantification of binding affinities. The umbrella sampling methodology has previously been largely limited to smaller systems, e.g. the binding of short peptides to proteins. In this study we have used coarse-grained molecular dynamics simulations and an umbrella sampling approach to quantify the binding of PH domains to model membranes containing PIP lipids, considering particularly the PH domain of GRP1. Out results show that the binding affinity of the GRP1-PH domain for PIP3 molecules in a lipid bilayer is greater than for PIP2, in line with previous studies, while other phospholipids did not exhibit favourable binding. Mutation of four key basic residues in the binding site reduced the affinity of GRP1-PH for PIP3. Analysis of the simulations shows that in addition to the canonical orientation, GRP1-PH can bind to the membrane in a secondary orientation via a separate PIP interaction site. This secondary site coincides with an ‘atypical’ site identified in several other PH domains. These results demonstrate how an umbrella sampling approach, combined with coarse-grained simulations, can be applied to peripheral protein-membrane systems in order to increase understanding of the energetics and molecular details of protein-lipid interactions.