Multivalent Membrane Lipid Targeting by the Calcium-Independent C2A Domain of Slp-4/Granuphilin

Abstract

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking insulin secretory vesicles to the plasma membrane prior to exocytosis. Slp-4 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). Its 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 multiple sites on the C2A domain. In addition to the previously identified, PIP-selective lysine cluster, a second cationic region of the protein domain contributes substantially to its affinity for physiologically relevant lipid compositions. Mutations at the two sites decrease affinity for PIP2 and background anionic lipids, respectively, while combined mutations at both sites are needed to block binding to physiologically relevant membranes. Docking and molecular dynamics simulations indicate that binding at the nonspecific site varies with protein conformation, suggest a possible allosteric mechanism of cooperativity between the two sites. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking to the plasma membrane.