The STXBP2 mutation does not influence protein expression or the Munc18-2/STX11 interaction. (A) Western blots showing the expression levels of Munc18-2, STX11, and MUNC13-4 in lysates prepared using PBMCs activated with CD3/28 beads from control (black) or P1 (red). Actin staining of the same membranes was used to assess for equivalent protein loading. (B) Bands in the western blot that corresponded to Munc18-2, STX11, and MUNC13-4 were quantified by densitometry and normalized to the intensity of actin in the same lane. Densitometry results are expressed as the percentage of those obtained using control samples, which were set as 100%. (C) Coimmunoprecipitation experiments using lysates generated from control or P1 PBMCs. Endogenous STX11 was immunoprecipitated (IP) using an anti-STX11 antibody, and the amount of Munc18-2 that coimmunoprecipitated was quantified by western blot analysis. (D) Bands in the western blot that corresponded to the fraction of Munc18-2 that coimmunoprecipitated (co-IP) with STX11 were quantified by densitometry and normalized to the amount of STX11 immunoprecipitated in the same lane. Densitometry results were expressed as the percentage of those obtained in control samples, which were set as 100%. (E) Coimmunoprecipitation experiments using HeLa cells transiently transfected with Myc-STX11 and ECFP-Munc18-2^WT (black) or ECFP-Munc18-2^R65Q (red). STX11 was immunoprecipitated (IP) using an anti-Myc antibody and the amount of Munc18-2 that coassociated was quantified by western blot analysis using an anti-GFP antibody. (F) Densitometry analysis was performed as described in panel C. In panels A-F, results are representative of 2 independent experiments. (G) Crystal structure of Munc18-2 (Protein Data Bank entry 4CCA) with the R65 residue highlighted in magenta. Previously described HLH-associated Munc18-2 missense mutants are highlighted in orange. (H) The crystal structure of Munc18-1 (green) in complex with STX1 (Habc domain: blue, H3 domain: cyan; Protein Data Bank entry 3C98) reveals that the C-terminal half of the STX1 H3 domain inserts deeply into the Munc18-1 central cavity. The R65 residue (highlighted in magenta) is in proximity to residues D242 and Y243 (yellow) within the STX1 H3 domain but it does not make direct contact with these residues. Munc18-1 residues corresponding to described missense mutations in Munc18-2 are highlighted in orange. The insets in G and H represent a higher magnification of the structure, rotated 90° clockwise in the x-axis.