Membrane Binding by Synaptotagmin-Like Protein 4: Site Directed Mutagenesis of the Lipid Interaction Surface

Abstract

Membrane-binding proteins play crucial roles in many biological process such as vesicle trafficking and exocytosis. Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector protein whose overexpression is known to suppress insulin secretion despite increasing the number of secretory vesicles docked to the plasma membrane. Therefore, its structural mechanism may hold clues for understanding the regulation of efficiency in exocytosis signaling. Slp-4 binds to vesicular Rab effector proteins through its N-terminal Slp homology domain, to SNARE complexes via its central linker region, and contains C-terminal tandem C2A and C2B domains that interact with plasma membrane lipids. We have previously shown that the Slp-4 C2A domain binds with high affinity to liposomes approximating the lipid composition of the plasma membrane interior leaflet. The C2A domain has a lysine cluster that binds to phosphatidylinositol-(4, 5)-bisphosphate (PIP2) and soluble inositol phosphates with micromolar affinity, but much more strongly to membranes containing both PIP2 and anionic background lipids such as phosphatidylserine (PS). We are combining computational and experimental approaches to reveal which residues of the Slp-4 C2A domain participate in binding PS and PIP2. Using mutational analysis and protein binding assays, here we show that mutation of specific individual amino acid residues tends to have modest effects on the domain's membrane affinity; however, combined mutations at multiple predicted binding sites have greater effects. This finding indicates that the C2A domain interacts with plasma membrane lipids through multiple binding sites that act in a concerted manner to produce its high membrane affinity.