(A–C) Ribbon diagrams illustrating the structures of the Syt1-SNARE complex solved by NMR spectroscopy (A) or X-ray crystallography without (B) or with a bound complexin-1 fragment (C), which revealed the binding modes mediated by the polybasic region, the primary interface and the tripartite interface, respectively. The PDB accession codes for the structures are 2N1T, 5KJ7 and 5W5C, respectively. Syntaxin-1 is in yellow, synaptobrevin in red, SNAP-25 in green, complexin-1 in pink and the Syt1 C2B domain in gray, with bound Ca2+ ions shown as orange spheres. The side chains of several residues from the polybasic region are shown as dark blue (K313, K321, R322, K325 and K327) or cyan spheres (K324 and K326), those of R398,R399 in primary region II as purple spheres and those of E295 and Y338 in primary region I as pink spheres. (D–G) Close-up views of the polybasic (D), primary region II (E), primary region I (F) and tripartite (G) interfaces. All side chains are shown as stick models, with the same color used in (A–C) for the C2B domain. For the SNARE complex, nitrogen atoms are in blue, oxygen atoms in red and carbon atoms in green (SNAP-25), yellow (syntaxin-1) and pink (complexin-1 and synaptobrevin). The L387 and L394 residues are colored in brown in panel G. Selected side chains are labeled. Note that the structure determined by NMR spectroscopy was highly dynamic and panels (A,D) show just one member of this dynamic ensemble that illustrates the common key feature of the ensemble, namely the involvement of the residues colored in dark blue in binding to a polyacidic patch of the SNARE complex. (H) Superposition of 1H-15N TROSY-HSQC spectra of 2H,15N-IM-13CH3-C2B domain in the absence of Ca2+ and the presence of different concentrations of CpxSC as indicated by the color code (C2B concentrations gradually decreased from 32 to 12 μM). Cross-peaks broadened gradually with increasing CpxSC concentrations and contour levels were adjusted to allow observation of most cross-peaks in each spectrum. Some cross-peaks broadened beyond detection at high CpxSC concentrations. (I) Chemical shift changes induced by 51 μM CpxSc on the 1H-15N TROSY-HSQC cross-peaks of the C2B domain. Composite Δδ values were calculated as [(Δδ1H)2+(0.17*Δδ15N)2]1/2, where Δδ1H is the chemical shift change in the 1H dimension and Δδ15N is the chemical shift change in the 15N dimension. (J) Summary of the largest chemical shift changes caused by 51 μM CpxSc on the 1H-15N TROSY-HSQC cross-peaks of the C2B domain. A ribbon diagram of the C2B domain (gray) with a SNARE complex bound to the primary interface on the left (light pink) and SNARE complex bound to the polybasic region on the right (light blue) is shown. C2B domain residues corresponding to cross-peaks with Δδ > 0.02 ppm (blue line in panel I) are highlighted in blue (polybasic region) or pink (primary interface). (K) Expansions showing the changes observed at increasing CpxSC concentrations in the cross-peaks corresponding to K325 and K369 at the polybasic region, and V283 and Y339 at the primary interface. Only spectra at selected concentrations of CpxSC are shown. The color code is the same as in panel H.