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

被引:14
作者
Alnaas, Aml A. [1 ]
Watson-Siriboe, Abena [1 ]
Tran, Sherleen [1 ]
Negussie, Mikias [1 ]
Henderson, Jack A. [1 ]
Osterberg, J. Ryan [1 ]
Chon, Nara L. [1 ]
Harrott, Beckston M. [1 ]
Oviedo, Julianna [1 ]
Lyakhova, Tatyana [1 ]
Michel, Cole [2 ]
Reisdorph, Nichole [2 ]
Reisdorph, Richard [2 ]
Shearn, Colin T. [3 ]
Lin, Hai [1 ]
Knight, Jefferson D. [1 ]
机构
[1] Univ Colorado, Dept Chem, Denver, CO 80202 USA
[2] Univ Colorado, Dept Pharmaceut Sci, Sch Med, Anschutz Med Campus, Aurora, CO USA
[3] Univ Colorado, Dept Pediat, Div Pediat Gastroenterol Hepatol & Nutr, Sch Med, Anschutz Med Campus, Aurora, CO USA
关键词
PARTICLE MESH EWALD; MOLECULAR-DYNAMICS; PLASMA-MEMBRANE; INSULIN GRANULES; GRANUPHILIN; BINDING; EXOCYTOSIS; SIMULATIONS; PARAMETERS; INSIGHTS;
D O I
10.1074/jbc.RA120.014618
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
070307 [化学生物学]; 071010 [生物化学与分子生物学];
摘要
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.
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页数:17
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