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  • Mammalian CAPS includes two i http www apexbt com

    2022-01-07

    Mammalian CAPS includes two isoforms, CAPS-1 and CAPS-2. Both isoforms contain several functional domains, including a C2 domain that is likely responsible for CAPS dimerization (Petrie et al., 2016), a pleckstrin homology (PH) domain that binds PIP2 (phosphatidylinositol 4,5-bisphosphate) and mediates the interaction of CAPS with the plasma membrane (Grishanin et al., 2002, Grishanin et al., 2004), and a C-terminal region that exhibits sequence homology to the Munc13 MUN domain (Koch et al., 2000, Pei et al., 2009, James and Martin, 2013). This C-terminal region of CAPS contains two adjacent helical fragments, DUF1041 (domain of unknown function 1041, pfam 06292) and MHD1 (Munc13 homology domain-1), which are referred to as the DAMH domain (residues 859–1073) (Khodthong et al., 2011). Recent studies have suggested that the DAMH domain constitutes the core SNARE-binding region of CAPS-1, which is essential for CAPS-1 function in SNARE-mediated liposome fusion and DCV GM6001 (Khodthong et al., 2011). However, no three-dimensional structure of the DAMH domain has been reported, hindering our understanding of the molecular mechanism of CAPS in SNARE complex formation and priming of exocytosis. Here we report a crystal structure of the rat CAPS-1 DAMH domain at 2.9-Å resolution. By virtue of combined in vitro and in vivo experiments, our data revealed two distinct roles of the DAMH domain that might be crucial for the function of CAPS-1 in SNARE complex formation: an inhibitory role dependent on binding of the DAMH domain to the MUN domain of Munc13-1, which hinders Munc13-catalyzed opening of Syx1, and a chaperone role dependent on interaction of the DAMH domain with the Syx1/SN25 complex, which stabilizes the open conformation of Syx1, facilitating SNARE complex formation. These results explain the complementary functions of CAPS-1 and Munc13-1 in priming of exocytosis, suggesting that CAPS-1 facilitates SNARE complex formation in a manner dependent on sequential and cooperative interaction with Munc13-1 and the SNARE proteins.
    Results
    Discussion The CAPS and Munc13 proteins promote SNARE-mediated liposome fusion and priming of DCVs and SVs during exocytosis, suggesting their essential role in facilitating trans-SNARE complex formation in advance or at the time of Ca2+-triggered exocytosis (James and Martin, 2013). CAPS and Munc13 function in a nonredundant manner, but the spatial and temporal regulation of these two factors during exocytosis remains unclear. The binding of CAPS-1 and Munc13-1 to the SNAREs is essential for their function in exocytosis. The principal regions responsible for SNARE binding in CAPS-1 and Munc13-1 were identified previously as the DAMH domain and the MUN domain, respectively (Khodthong et al., 2011, Ma et al., 2011). In this study, we reported the crystal structure of the DAMH domain of CAPS-1 and explored the sequential and cooperative actions of CAPS-1 and Munc13-1 in SNARE complex formation. Critical properties of the DAMH domain of CAPS-1 that are relevant to priming of exocytosis have been identified, adding to our understanding of the mechanism of CAPS-1 in exocytosis. Compared with the MUN domain (Yang et al., 2015), the DAMH domain exhibits a similar elongated structure that consists of stacked α helices but adopts a more bent architecture and contains more negatively charged residues on the outer surface. CAPS-1 binds membrane-associated Syx1/SN25 complexes, with Syx1 and SN25 each contributing a binding site for the DAMH domain (Daily et al., 2010, Khodthong et al., 2011). Among these interactions, the sites identified for DAMH domain binding in Syx1 are located in the membrane-proximal linker region that connects the SNARE motif and the transmembrane domain, including residues K252, K260, and K265 (Daily et al., 2010). Consistent with this finding, three negatively charged patches (E890-H891-E894, W898-D901-L902, and E905) located in helix H1 of the DAMH domain were identified to be essential for binding to the Syx1/SN25 complexes (Figure 4), and mutation of these residues impairs CAPS-1 activity in SNARE-mediated fusion and evoked DCV exocytosis in PC12 cells (Figures 5 and 6). These results correlate well with the previously observed fusion defects caused by the K260Q-K265L mutations in the membrane-proximal linker region of Syx1 (Daily et al., 2010), suggesting a charge-charge binding mode between CAPS-1 and the SNAREs. Our in vitro and in vivo data strongly support the notion that CAPS-1 functions in DCV exocytosis in a manner dependent on an interaction between the DAMH domain and the Syx1/SN25 complex. Based on the structural data and functional results, helix H1 of the DAMH domain appears to represent the principal region that mediates SNARE binding. Interestingly, the membrane-proximal linker region of Syx1 also mediates binding of the Munc18-1/Syx1 complex to the DAMH domain (Figure S5), suggesting that the same sequence of Syx1 provides a consistent binding target for the DAMH domain regardless of whether it is within the Munc18-1/Syx1 complex or within the Syx1/SN25 complex.