High affinity interactions of Pb ²⁺ with synaptotagmin I

Lead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca ²⁺ -evoked neurotransmitter release, has two high-affinity Pb ²⁺ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb ²⁺ -complexed C2 domains revealed that protein-bound Pb ²⁺ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb ²⁺ binding to the C2 domains of SytI are comparable to those of Ca ²⁺ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb ²⁺ can serve as both a thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb ²⁺ ions inhibits further Ca ²⁺ 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 Pb ²⁺ ions can interfere with the Ca ²⁺ -dependent function of SytI in the cell.