Membrane Docking of the Synaptotagmin 7 C2A Domain: Computation Reveals Interplay between Electrostatic and Hydrophobic Contributions

Abstract

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.

The Effects of Ca2+ Concentration and E200K Mutation on the Aggregation Propensity of PrPC: A Computational Study

“…Parallely, it is worth taking into consideration that a maximum response in either cytotoxicity and/or conductance assays have been analogously gained for [Ca 2+ ] > 5 mM [17], thus suggesting that the formation of DBM itself may have some functional meaning. Indeed, the DBM detected in region 1 of PrP C showed appreciable similarities with multinuclear Ca 2+ binding sites found in other proteins such as thermolysin [36, 37], synaptotagmin [38], and perforin [39]. Therefore, further corroboration to the DBM formation was provided by quantum mechanical calculations that substantially confirmed the coordination geometry obtained by MD calculations.…”

The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis

“…Biochemical experiments and atomistic molecular dynamics simulations demonstrate a large hydrophobic component to C2A lipid interactions that plays a role in increasing binding stability. A significant contribution to the energetic stabilization arises from the phenylalanine residues at the tips of Ca 2+ binding loops 1 and 3 including Phe167, which corresponds to a methionine in Syt-1 ( Brandt et al, 2012 ; Chon et al, 2015 ; Vermaas and Tajkhorshid, 2017 ). Recently, a second important residue was also identified in the Syt-7 C2A binding loop: Arg231, whose forked guanidino group binds more efficiently to PS than the lysine found at the corresponding position in Syt-1.…”

“…6 ) orients toward the plasma membrane and binds PIP2, whereas the polybasic region of C2A orients toward the vesicle. Although the polybasic region of synaptotagmin C2A domains is not particularly selective for PIP2 binding because of the presence of a glutamate residue at a key position ( Guillén et al, 2013 ), it is nevertheless present in Syt-1 as a region of positive surface charge density and has a greater surface potential in Syt-7 ( Chon et al, 2015 ). The model in Fig.…”

Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane

“…The PC:SM:CHOL bilayer was prepared using the CHARMM-GUI (47), shortly equilibrated (500 ps) in the NVT ensemble and simulated further during 500 ns in the NPT ensemble. The PC:PS bilayer was taken from Chon et al (48). The PC:PI(4)P bilayer was also built using the CHARMM-GUI and was simulated for 200 ns in the NPT ensemble after a short NVT equilibration of 500 ps.…”