Fig. 4.

Structure and membrane interactions of the Synaptotagmin‐1 C₂ domains. (A, B) Ribbon diagrams of the NMR structures of the Syt1 C₂A (A) and C₂B (B) domains [47, 51] bound to Ca²⁺ (orange spheres) (PDB accession numbers 1BYN and 1K5W, respectively) in the approximate orientations with respect to a flat phospholipid bilayer (gray) defined by EPR [105, 107]. The slightly slanted orientation of the C₂B domain in (B) enables interactions of residues from the polybasic region with PIP₂ head groups that protrude from the bilayer surface (red hexagon). Basic residues involved in SNARE complex and membrane interactions are labeled and shown by stick models. Hydrophobic side chains that insert into the membrane are shown as green stick models. Note that K313, R322, K325 and K327 in the polybasic face of the C₂B domain can readily bind to PIP₂ in these orientations whereas K324 and K326 are oriented away from the membrane, farther from the PIP₂ head group. These observations explain the selective disruption of neurotransmitter release caused by mutations in the polybasic face [108, 112]. (C) Approximately parallel orientation of the C₂B domain with respect to the membrane expected in the absence of Ca²⁺, which is supported by MD simulations [87]. (D–F) Selectively strong disruption of Syt1 C₂AB binding to membranes by the R322E,K325E (REKE) mutation but not by the K324E,K326E (KEKE) mutation is observed in the presence of Ca²⁺ and PIP₂ but not in the absence of Ca²⁺ and/or PIP₂. The plots show binding of C₂AB to nanodiscs containing PS and PIP₂ (D, E) or PS but not PIP₂ (F) in the presence of Ca²⁺ and 125 mm KCL (D, F) or EGTA and 50 mm KCl (E). These data were reported in [45]. The data in panel (E) were acquired at lower ionic strength because binding is very weak in the absence of Ca²⁺.