Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin

J Biol Chem. Jan-Jun 2021;296:100159. doi: 10.1074/jbc.RA120.014618. Epub 2020 Dec 10.


Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology 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). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. 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.

Keywords: C2 domain; MIN6; Slp4; electrostatics; granuphilin; insulin secretion; membrane binding.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line, Tumor
  • Cholesterol / chemistry*
  • Cholesterol / metabolism
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Humans
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / metabolism
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism
  • Liposomes / chemistry*
  • Liposomes / metabolism
  • Mice
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Phosphatidylcholines / chemistry*
  • Phosphatidylcholines / metabolism
  • Phosphatidylethanolamines / chemistry
  • Phosphatidylethanolamines / metabolism
  • Phosphatidylinositol 4,5-Diphosphate / chemistry*
  • Phosphatidylinositol 4,5-Diphosphate / metabolism
  • Phosphatidylinositols / chemistry
  • Phosphatidylinositols / metabolism
  • Phosphatidylserines / chemistry
  • Phosphatidylserines / metabolism
  • Protein Binding
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sphingomyelins / chemistry
  • Sphingomyelins / metabolism
  • Vesicular Transport Proteins / chemistry*
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism


  • Lipid Bilayers
  • Liposomes
  • Phosphatidylcholines
  • Phosphatidylethanolamines
  • Phosphatidylinositol 4,5-Diphosphate
  • Phosphatidylinositols
  • Phosphatidylserines
  • Recombinant Proteins
  • SYTL4 protein, human
  • Sphingomyelins
  • Vesicular Transport Proteins
  • 1-palmitoyl-2-oleoylglycero-3-phosphoserine
  • Cholesterol
  • 1-palmitoyl-2-oleoylphosphatidylcholine
  • 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine