Critical residues in munc13-4 required for rab27 binding. (A) Organization of munc13-4 and truncations used in this study. (B) Binding assay of ³⁵S-labeled His₆munc13-4 truncations and GSTrab27aGTPγS. Bound proteins were resolved on a 10% SDS-PAA gel and visualized by phosphor imaging. (C) FLAG-tagged rab27a and GFP-munc13-4 truncations were coexpressed in COS-7 cells. Anti-FLAG immunoprecipitates were analyzed by Western blot with a GFP mAb. (D) Binding assay of ³⁵S-labeled His₆munc13-4 AAA mutants and GSTrab27aGTPγS. Bound protein was eluted and assayed by phosphor imaging. The amino acid sequence containing the R27BD is given at the bottom of the panel, and the coloring of the blocks indicates the percentage binding as per the legend in the panel. (E) Model of the 3D structure of the C2A domain of munc13-4 (aa 111-284, ribbon diagram). The model was made on the basis of the alignment⁸  of the C2A domain of human munc13-4 with the C2B domain of rat munc13-1, for which the experimental 3D structure (pdb 3kwu) has been solved.²⁶  The limits of the 240-284 segment, which includes the β-strands βA to βC, are shown, as well as the amino acids discussed in the text. Loops that were not modeled are symbolized with dashed lines. Amino acids of the Ca²⁺-binding site are shown, together with the bound Ca²⁺ ions (green spheres). (F) Binding assay of ³⁵S-labeled His₆munc13-4Δ (280-285) or His₆munc13-4 and GSTrab27aGTPγS. Bound protein was eluted and assayed by phosphor imaging. Quantitations of signals present in bound and total samples and background correction were done with the ImageQuant software package.