The AD3 locus of synaptotagmin-1 C2 domains modulates domain stability

Synaptotagmin-1 (syt1) functions as the Ca²⁺-dependent sensor that triggers the rapid and synchronous release of neurotransmitters from neurotransmitter-containing vesicles during neuronal exocytosis. The syt1 protein has two homologous tandem C2 domains that interact with phospholipids in a Ca²⁺-dependent manner. Despite the crucial role of syt1 in exocytosis, the precise interactions between Ca²⁺, syt1, and phospholipids are not fully understood. In a study involving recessive lethal mutations in the syt1 gene, a specific mutation named AD3 was generated in Drosophila syt1, resulting in a significant reduction in Ca²⁺-dependent exocytosis. Further investigation revealed that the AD3 mutation was a missense mutation located in a conserved consensus sequence within the C2B domain of Drosophila syt1. However, the biophysical impact of the AD3 mutation had not been analyzed. Our study uses x-ray crystallography, isothermal titration calorimetry, thermodynamic analysis, and molecular dynamics simulation to show that the primary defect caused by the AD3 mutation in the syt1 protein is reduced thermodynamic stability. This instability alters the population of Ca²⁺-receptive states, leading to two major consequences: decreased affinity for calcium ions and compromised stabilization of the domain normally enhanced by Ca²⁺. We conclude that this conserved residue acts as a structural constraint, delimiting the movement of loop 3 within the pocket and ultimately influencing the affinity of the calcium ion binding with the C2 domain.