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.
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D050863:Synaptotagmin I; C424135:Sytl3 protein, mouse; D050857:Synaptotagmins; D002118:Calcium