Binding kinetics and ligand specificity for the interactions of the C2B domain of synaptogmin II with inositol polyphosphates and phosphoinositides.
Synaptotagmin II (Syt II) is a key protein in the calcium-dependent exocytosis of synaptic vesicles. It contains two domains homologous to the C2 regulatory region of protein kinase C. The C2A domain acts as a calcium sensor, while the C2B domain has high affinity for inositol polyphosphates (InsP(n)()s) and phosphoinositide polyphosphates (PtdInsP(n)()s). We describe the use of a surface plasmon resonance biosensor in determining the binding kinetics of the C2B domain with InsP(n)() and PtdInsP(n) ligands. Biosensor surfaces were prepared with covalently attached Ins(1,4,5)P(3), Ins(1,3,4,5)P(4), and InsP(6) ligands. The interactions of bacterially expressed His(6)-tagged C2B and (C2A+C2B) domains of Syt II were examined in the presence and absence of competing InsP(n)s and PtdInsP(n)s. Both His(6)-C2B and His(6)-(C2A+C2B) exhibited the highest affinity for the Ins(1,3,4,5)P(4)-modified surface with a K(D) value of 6 nM. The His(6)-(C2A+C2B) had a 10-fold lower association rate constant for the InsP(6)-linked surface (k(a) = 4.6 x 10(3) M(-1) s(-1)) than for the Ins(1,3,4,5)P(4)-modified surface (k(a) = 6.8 x 10(4) M(-1) s(-1)). Two water-soluble phosphoinositides, dioctanoyl-PtdIns(3,4,5)P(3) and dioctanoyl-PtdIns(4,5)P(2), were superior to the soluble InsP(n)s in displacing binding to the Ins(1,3,4,5)P(4)-modified surface. The binding of His(6)-C2B and His(6)-(C2A+C2B) to InsP(n) surfaces did not show significant calcium dependence. These data support a model in which the binding of the C2B domain of Syt II to PtdInsP(n)s is important for the docking and/or fusion of the secretory vesicles to the synaptic plasma membrane.
Department of Medicinal Chemistry, The University of Utah, 30 South 2000 East, Room 201, Salt Lake City, Utah 84112-5820, USA.
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Mehrotra
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Myszka
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D001667:Binding, Competitive; D015374:Biosensing Techniques; D002118:Calcium / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008962:Models, Theoretical; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010833:Phytic Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050861:Synaptotagmin II
D007295:Inositol Phosphates; D008024:Ligands; D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; D050861:Synaptotagmin II; C081655:inositol-1,3,4,5-tetrakisphosphate; D010833:Phytic Acid; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium