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 exocyto sis of synaptic vesicles. It contains two domains homologous to the C2 regu latory 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 kinetic s 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 Hiss-tagged C2B and (C2A+C2B) domains of Syt LI 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 wit h a K-D value of 6 nM. The His(6)-(C2A+C2B) had a 10-fold lower association rate constant for the LnsP(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 watersoluble phosphoinositides, dioctanoyl-PtdIns(3,4,5)P-3 and di octanoyl-PtdIns(4,5)P-2, were superior to the soluble InsP(n)s in displacin g binding to the Ins(1,3,4,5)P-4-modified surface. The binding of His(6)-C2 B and His(6)-(C2A+C2B) to InsP(n) surfaces did nor show significant calcium dependence. These data support a model in which the binding of the C2B dom ain of Syt II to PtdInsP(n)s is important for the docking and/or fusion of the secretory vesicles to the synaptic plasma membrane.