title: The C2B domain of synaptotagmin I is a Ca2+-binding module.
abstract: Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.
authors.affiliation: Department of Biochemistry, Center for Basic Neuroscience, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ubach
authors.forename: Y
authors.initials: Y
authors.lastname: Lao
authors.forename: I
authors.initials: I
authors.lastname: Fernandez
authors.forename: D
authors.initials: D
authors.lastname: Arac
authors.forename: T C
authors.initials: TC
authors.lastname: Südhof
authors.forename: J
authors.initials: J
authors.lastname: Rizo
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D019281:Dimerization; D004340:Drug Contamination; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D011107:Polylysine; D002118:Calcium