Non-Native Metal Ions Reveal The Role Of Electrostatics In Synaptotagmin 1-Membrane Interaction

Synaptotagmin 1 (Syt1) belongs to the family of evolutionarily conserved C2 domain proteins that are named after the second conserved domain of protein kinase C (PKC). The two C2 domains of Syt1 (designated as C2A and C2B) act as Ca2+ sensors for the evoked synchronous neurotransmitter release. The binding of Ca2+ ions to the tip of Syt1 C2 domains changes the local electrostatic potential from positive to negative. This “electrostatic switch” is believed to be sufficient to allow the protein to interact with anionic phospholipids in the presynaptic membranes, a function essential for synaptic vesicle fusion. Non-native toxic metal ions such as Cd2+ and Pb2+ can potentially bind to the C2 domains as Ca2+ mimics and interfere with this critical function. The goal of our research is to determine how the binding of such toxic metals affects the structure and function of Syt1. Using solution NMR spectroscopy and the lanthanide (Tb3+) luminescence experiments, we determined that C2 domains have a higher affinity for Cd2+ and Pb2+ than the native ligand, Ca2+. We were able to obtain high-resolution crystal structures of both C2 domains of Syt1 complexed to Cd2+ and Pb2+ and identify the high-affinity sites. We also demonstrate that although Cd2+ binds to Syt1 with high affinity, it fails to support the protein-membrane interactions. In contrast, Pb2+ acts as a functional surrogate of Ca2+ in driving Syt1-membrane association. This seemingly contradictory behavior suggests that the simple “electrostatic switch” model does not accurately describe Syt1-membrane interactions. The absence of membrane binding with Cd2+ bound C2 domains could be due to the inability of Cd2+ to effectively coordinate the oxygen atoms of anionic phospholipid head groups. Our data supports the notion that the direct coordination of the protein-bound metal ion by anionic lipid head group is essential for the membrane binding of Syt1. Acknowledgement: This research is supported by the funds from Welch Foundation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 GM108998.