Multivalent Membrane Lipid Targeting by the Calcium-Independent C2 Domains of Granuphilin: Evidence from Computation and Experiment

Abstract

Granuphilin, also known as synaptotagmin-like protein 4 (Slp4), is a Rab effector responsible for docking insulin secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, binds plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). Granuphilin's C2A domain has previously been shown to bind PIP2 or its soluble analogues with low micromolar affinity; however, the domain docks with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS). Here we show using a combination of computational and experimental approaches that this high-affinity membrane interaction arises from concerted binding at two or more sites on the C2A domain. First, docking calculations predict at least two possible binding pockets for anionic ligands. Second, liposome binding measurements indicate that affinity depends on concentrations of PIP2 as well as PS and phosphatidic acid (PA) in the membrane. Third, fluorescence measurements indicate that different regions of the protein surface are responsible for binding membranes containing or lacking PIP2. Fourth, single-molecule lateral diffusion measurements indicate distinct membrane-bound states depending on the available target lipids. Mutational analysis is currently underway to confirm locations of these lipid binding sites. Overall, multivalent membrane binding by granuphilin likely contributes to selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.