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. 2015 Sep 22;54(37):5684-95.
doi: 10.1021/acs.biochem.5b00421. Epub 2015 Sep 10.

Membrane Docking of the Synaptotagmin 7 C2A Domain: Electron Paramagnetic Resonance Measurements Show Contributions from Two Membrane Binding Loops

J Ryan Osterberg  1 , Nara Lee Chon  1 , Arthur Boo  1 , Favinn A Maynard  1 , Hai Lin  1 , Jefferson D Knight  1
Affiliations

Membrane Docking of the Synaptotagmin 7 C2A Domain: Electron Paramagnetic Resonance Measurements Show Contributions from Two Membrane Binding Loops

J Ryan Osterberg et al. Biochemistry. .

Abstract

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, at least in part because of greater contributions from the hydrophobic effect. While the structure and membrane activity of Syt1 have been extensively studied, the structural origins of differences between Syt1 and Syt7 are unknown. This 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 the Syt1 C2A domain. 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 1 (CBL1) and 3 (CBL3), and membrane binding is more sensitive to mutations in CBL3. On average, Syt7 C2A inserts more deeply into 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.

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Figures

Figure 1
Figure 1
Spin labeling of Syt7 C2A. A: Solution NMR structure of Syt7 C2A (PDB ID: 2D8K). Yellow spheres indicate residues selected for spin labeling in this study. B: Structure of the MTSSL tag, with dihedral angles χ1 – χ5 indicated.
Figure 2
Figure 2
Bound and unbound EPR spectra for each spin-labeled mutant. Bound spectra (red) were measured with 30–150 μM protein and 30 mM (3:1 PC:PS) lipid membrane concentration with 1.5 – 2.0 mM Ca2+. Unbound spectra (blue) were measured with similar protein concentrations but in free solution with identical Ca2+ concentration. Signal broadening is indicative of a less mobile spin label.
Figure 3
Figure 3
Hyperbolic tangent fits of experimentally determined Syt7 C2A depth parameters. Open circles indicate doxyl lipids, solid black circles indicate single-cysteine mutants. Each fit is based on Equation 7–8. Error bars are 95% CI of depth parameter values measured in duplicate or triplicate, which are listed in Table 2. The same depth parameter data were used for each fit, and applied to (A) Ca2+-bound structure A, (B) Ca2+-bound structure B, (C) Ca2+-bound structure C, (D) NMR structure state 1, or (E) NMR structure state 2. The relevant NMR structure is PDB ID 2D8K.
Figure 4
Figure 4
Optimized best-fit docking models for Syt7 C2A based on the indicated starting structure. Black lines represent the lipid phosphate plane, blue shading represents the approximate headgroup region and yellow shading represents the approximate nonpolar acyl chain region. The phenylalanine residues at positions 167 and 229 are shown in green, and bound Ca2+ ions are shown in orange. Protein orientations correspond to successive Euler angle rotations of θx and θz followed a by translation along the y axis of ytrans, as follows: Structure A: θx = 252.8°, θz = −4.4°, ytrans = −18.2 Å; Structure B: θx = 268.6°, θz = 11.1°, ytrans = −16.8 Å; Structure C: θx = 261.8°, θz = 14.7°, ytrans = −17.1 Å; 2D8K State 1: θx = 17.5°, θz = 4.8°, ytrans = −20.6 Å; 2D8K State 2: θx = 23.1°, θz = −6.4°, ytrans = −22.2 Å. Starting coordinates for Structures A, B, and C are available in the Supporting Information.
Figure 5
Figure 5
Comparison of (A) the median Syt7 C2A docking geometry determined here (Structure B in Figure 4) with (B) the reported Syt1 C2A docking geometry of the isolated domain and (C) the reported Syt1 C2A docking geometry in the C2AB tandem. Optimized docking geometries are superimposed on a simulated lipid bilayer containing 25% POPS and 75% POPC. Hydrophobic sidechains on CBL1 and CBL3 are shown in green, and Ca2+ ions are shown in orange. Syt1 C2A geometries are reconstructed based on the transformations reported previously.,
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