Dynamic Control of Ca2+ Binding in the C2 Domains of Synaptotagmin 1

Abstract

Synaptotagmin senses fluctuations in the Ca2+ environment of neurons near active zones and transduces a signal to the SNARE complex to initiate exocytosis at the presynaptic terminus. The 3D structures of the two tandem C2 domains of synaptotagmin have been determined to high resolution; however, it is currently unclear how each domain dynamically interacts with Ca2+ at the atomic level. To study the mechanistic consequences of the lethal mutations at the AD3 locus, we introduced tyrosine to asparagine point mutations in both the C2A and C2B domains of synaptotagmin 1, and we have constructed a model that describes the relationship between Ca2+ -binding and the structural changes within each C2 domain. We show that the mobility of loop 3 in the Ca2+ binding pocket increases markedly in C2A, while the mobility of loop 1 changes in C2B with the AD3 mutation. This increase in loop mobility results in an increase in the average volume and variance of the Ca2+ -binding pockets of C2A and C2B. The volume of the unbound Ca2+ -binding pocket in C2A is usually restrained by intra-domain interactions between the tyrosine residue at the AD3 locus and residues on loop 3; however, the AD3 mutation decouples the restraint and results in a larger, more variable Ca2+ -binding pocket in C2A. C2B maintains a more compact Ca2+ -binding pocket; however, its volume also fluctuates significantly with the AD3 mutation. Changes in binding pocket volume that involve more variable Ca2+ binding loops would likely affect Ca2+ affinity in the neurons of the affected organism. Using molecular-dynamics simulations, we show that mutations at the AD3 locus alter the mobility of the Ca2+ -binding loops by removing a key stabilization mechanism that is normally present in C2 domains. The lack of loop stabilization results in a net increase in the volume of the Ca2+ -binding pocket and provides an explanation for the observed lethal phenotype.