High affinity interactions of Pb2+ with synaptotagmin I

Abstract

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca²⁺-evoked neurotransmitter release, has two high-affinity Pb²⁺ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb²⁺-complexed C2 domains revealed that protein-bound Pb²⁺ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb²⁺ binding to the C2 domains of SytI are comparable to those of Ca²⁺ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb²⁺ can serve as both a thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb²⁺ ions inhibits further Ca²⁺ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(II) and suggests a mechanism by which low concentrations of Pb²⁺ ions can interfere with the Ca²⁺-dependent function of SytI in the cell.