The C2A domain of synaptotagmin-like protein 3 (Slp3) is an atypical calcium-dependent phospholipid-binding machine: comparison with the C2A domain of synaptotagmin I.
The synaptotagmin-like protein (Slp) family consists of an N-terminal Rab27- binding domain and C-terminal tandem C2 motifs, and although it has been suggested to regulate Rab27-dependent membrane trafficking, such as Ca2+-regulated granule exocytosis in T-lymphocytes [Kuroda, Fukuda, Ariga and Mikoshiba (2002) J. Biol. Chem. 277, 9212-9218], little is known about the Ca2+-binding property of the Slp family. In this study, I demonstrated that the C2A domain of Slp3 exhibits Ca(2+)-dependent phospholipid- binding activity similar to that of the C2A domain of synaptotagmin I (Syt I) with regard to phospholipid selectivity, bivalent cation selectivity and effect of ionic strength. This finding was surprising because the C2A domains of other C-terminal-type (C-type) tandem C2 proteins require five conserved acidic residues in the putative Ca2+-binding loops 1 and 3 on the top of the beta-sandwich structure for their Ca2+-/phospholipid-binding activity, whereas the C2A domain of Slp3 contains only one conserved acidic residue in the putative Ca2+-binding loop 1. Site-directed mutagenesis and chimaeric analysis of the C2A domains of Syt I and Slp3 showed that Glu-336 and Glu-337 in the putative Ca2+-binding loop 1 and polybasic sequence (Lys-359, Lys-360 and Lys-361) in the beta-4 strand of the C2 structure are crucial for Ca2+-dependent phospholipid- binding activity of the Slp3 C2A domain, whereas the similar polybasic sequence in the C2A domain of Syt I is dispensable for Ca2+-dependent phospholipid-binding activity. These results indicate that the C2A domain of Slp3 is an atypical Ca2+-/phospholipid-binding machine, compared with other C-type tandem C2 proteins.[1]References
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