Structural And Kinetic Characterization Of High-Affinity Lead(Ii)-Synaptotagmin I Interactions

Lead is an environmental toxin that is known to disrupt Ca2+-mediated signal transduction and neurotransmitter release. Although several Ca2+ sensor proteins involved in signaling are proposed as the molecular targets of Pb2+, how they are affected at the atomic level by Pb2+ exposure is not fully understood. Using biophysical techniques such as X-ray crystallography and NMR spectroscopy, we have identified distinct Pb2+ binding sites on the two Ca2+-binding C2 domains (named C2A and C2B) of Synaptotagmin I (SytI), a major Ca2+ sensor of evoked neurotransmitter release. Thermodynamic analysis of these sites using isothermal titration calorimetry revealed that the affinity of Pb2+ for the C2 domains exceeds those of the native ligand Ca2+ by 2-3 orders of magnitude. We used NMR ZZ exchange spectroscopy to determine the kinetics and activation parameters of Pb2+ association with the C2 domains. We found that although the on-rate constants for Pb2+ and Ca2+ both reflect a diffusion-limited binding, the off-rate constant for Pb2+ is two orders of magnitude slower than Ca2+. Furthermore, we observed that the C2 domains complexed with Pb2+ at the high-affinity sites have severely reduced affinity towards Ca2+, despite the existing coordination vacancies. This observation provides one of the potential mechanisms by which Pb2+ could disrupt the function of Ca2+ sensor proteins at concentrations several-fold lower than Ca2+. Taken together, our atomic level characterization of the SytI-Pb2+ association provides an insight into the potential modes of Pb2+ toxicity. [Acknowledgement: This research is supported by the funds from Welch Foundation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 GM108998.]