Intrinsically disordered regions of Src and Abl kinases contribute to membrane binding

Membrane targeting regulates protein localization and function. Abl and Src kinase feature an N-terminal myristoylation site followed by an intrinsically disordered region (IDR) of positive net charge, suggesting these regions contribute to membrane targeting. We investigated the interplay of myristoylation and IDR binding to phosphatidylinositol-4,5-bisphosphate (PIP<inf>2</inf>)-containing membranes using atomistic equilibrium and non-equilibrium molecular dynamics (MD) simulations. After having established Charmm36m-TIP4P-fb as a force field combination of choice for the challenging IDR-membrane systems of interest, we assessed the binding propensities of Abl and Src’s IDRs to lipid bilayers, and observed strong binding only when PIP<inf>2</inf> was present, as also quantified by significant increases in detachment forces and unbinding free energies when including 10 mol% PIP<inf>2</inf>. Interactions were mediated by saltbridges between the IDR’s basic residues and PIP<inf>2</inf> headgroups, and largely unaffected by cholesterol. On this basis, we designed Src and Abl mutants with deficient PIP<inf>2</inf> binding, and confirmed impaired membrane binding using liposomal-binding assays and atomic force microscopy experiments. Simulations and experiments of further variants corroborated that both a myristoylation site and the charged IDR are required for firm PIP<inf>2</inf>-membrane interactions. Our results establish myristoylated IDRs in kinases - and potentially beyond - as important regulators of PIP<inf>2</inf>-specific membrane targeting.