Multivalent 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, interacts with 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 interaction at multiple sites on the C2A domain. In addition to the previously identified, PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. While mutations at the PIP2-selective site decreases affinity for PIP2, multiple mutations are needed to decrease binding to physiologically relevant lipid compositions. Docking and molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. In addition, we identify and characterize a covalently modified variant in the bacterially expressed protein, which arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and has a low membrane affinity. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

INSTRUMENT(S): 6520A Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Cole Michel  

LAB HEAD: Cole Robert Michel

PROVIDER: PXD021393 | Pride | 2020-12-08

REPOSITORIES: Pride