The second C2-domain of copine-2, copine-6 and copine-7 is responsible for their calcium-dependent membrane association.

The copine family of proteins contains nine members with a similar domain structure, namely two N-terminal C2-domains (C2A and C2B) and a C-terminal A-domain. The former are thought to be responsible for binding to the inner face of the plasma membrane following increases in intracellular calcium levels, whereas the A-domain has been suggested to be a protein-binding structure. In this study, we examined the effects of mutagenesis of selected residues in the linker area between the C2-domains and the A-domain, and mutagenesis of the aspartates of the C2-domains, which are predicted to bind calcium and promote membrane association of the copines. We found that Lys282-Lys284 of the linker area are important for the folding of the intact protein. We showed that substitution with asparagine, single or multiple, of the aspartates in the C2A-domain had no effect on the calcium-mediated membrane association of copine-2, copine-6, or copine-7. Similar mutagenesis of a single residue in the C2B-domain of copine-6 (but not copine-2 and copine-7) was sufficient to eliminate its calcium-mediated membrane binding, and simultaneous substitution of all four of the asparagines in the C2B-domain resulted in constitutive membrane association of copine-2, copine-6 and copine-7 with the plasma membrane. These data show that the C2B-domains of copine-2, copine-6 and copine-7 are the domains responsible for the protein calcium-dependent membrane association.

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