This study provides novel mechanistic insights into how Syt-1 and Syt-7 C2B domains regulate the later stages of exocytosis. We show that small structural differences within this region exert significant effects on fusion pore properties. We propose that these effects may arise from differences in C2B domain affinity for the plasma membrane.

MacDougall et al. review the structure and function of the calcium sensor synaptotagmin-7 in exocytosis.

The synaptotagmin (Syt) family of proteins plays an important role in vesicle docking and fusion during Ca 2+ -induced exocytosis in a wide variety of cell types. Its role as a Ca 2+ sensor derives primarily from its two C2 domains, C2A and C2B, which insert into anionic lipid membranes upon binding Ca 2+ . Syt isoforms 1 and 7 differ significantly in their Ca 2+ sensitivity; the C2A domain from Syt7 binds Ca 2+ and membranes much more tightly than the C2A domain from Syt1, due at least in part to greater contributions from the hydrophobic effect. While the structure and membrane activity of Syt1 have been extensively studied, the structural origins of differences between Syt7 and Syt1 are unknown. The present study used site-directed spin labeling and electron paramagnetic resonance spectroscopy to determine depth parameters for the Syt7 C2A domain, for comparison to analogous previous measurements with Syt1 C2A. In a novel approach, the membrane docking geometry of both Syt1 and Syt7 C2A was modeled by mapping depth parameters onto multiple molecular dynamics simulated structures of the Ca 2+ -bound protein. The models reveal membrane penetration of Ca 2+ binding loops (CBLs) 1 and 3, and membrane binding is more sensitive to mutations in CBL3. On average, Syt7 C2A inserts more deeply in the membrane than Syt1 C2A, although depths vary among the different structural models. This observation provides a partial structural explanation for the hydrophobically driven membrane docking of Syt7 C2A. Keywordsprotein-lipid interaction; membrane bound structure; calcium signaling; exocytosis; power saturation Synaptotagmins (Syt) are a family of proteins characterized by their membrane targeting C2 domains. Syt proteins contain a transmembrane helix which anchors a cytoplasmic region consisting of two C2 domains (C2A and C2B) connected by a short linker. 1, 2 Each C2 domain is composed of two four-stranded β-sheets forming a β-sandwich structure with three flexible Ca 2+ binding loops, although not all Syt C2 domains bind Ca 2+ . 3,4 There are 17 human isoforms of Syt, eight of which show varying degrees of Ca 2+ binding affinity. 5 HHS Public Access Author manuscriptBiochemistry. Author manuscript; available in PMC 2016 September 22. Published in final edited form as:Biochemistry. 2015 September 22; 54(37): 5684-5695. doi:10.1021/acs.biochem.5b00421. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe individual C2 domains of each Syt exhibit a variety of affinities; for example, Syt1 C2A binds anionic lipids such as phosphatidylserine (PS) nonspecifically in the presence of Ca 2+ , while Syt1 C2B additionally binds phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in a partially Ca 2+ -independent fashion. [8][9][10][11][12] Syt1 C2B may additionally interact with the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) protein SNAP25 in a manner proposed to inhibit vesicle fusion prior to Ca 2+ influx. [13][14][15] Influx of Ca 2+ into the cytoplasm releases the fus...

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 (PIP 2 ). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP 2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP 2 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 PIP 2 -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 PIP 2 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 PIP 2 -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.

The C2A domain of synaptotagmin 7 (Syt7) is a Ca(2+) and membrane binding module that docks and inserts into cellular membranes in response to elevated intracellular Ca(2+) concentrations. Like other C2 domains, Syt7 C2A binds Ca(2+) and membranes primarily through three loop regions; however, it docks at Ca(2+) concentrations much lower than those required for other Syt C2A domains. To probe structural components of its unusually strong membrane docking, we conducted atomistic molecular dynamics simulations of Syt7 C2A under three conditions: in aqueous solution, in the proximity of a lipid bilayer membrane, and embedded in the membrane. The simulations of membrane-free protein indicate that Syt7 C2A likely binds three Ca(2+) ions in aqueous solution, consistent with prior experimental reports. Upon membrane docking, the outermost Ca(2+) ion interacts directly with lipid headgroups, while the other two Ca(2+) ions remain chelated by the protein. The membrane-bound domain was observed to exhibit large-amplitude swinging motions relative to the membrane surface, varying by up to 70° between a more parallel and a more perpendicular orientation, both during and after insertion of the Ca(2+) binding loops into the membrane. The computed orientation of the membrane-bound protein correlates well with experimental electron paramagnetic resonance measurements presented in the preceding paper ( DOI: 10.1021/acs.biochem.5b00421 ). In particular, the strictly conserved residue Phe229 inserted stably ∼4 Å below the average depth of lipid phosphate groups, providing critical hydrophobic interactions anchoring the domain in the membrane. Overall, the position and orientation of Syt7 C2A with respect to the membrane are consistent with experiments.