Chasing the Functional Asymmetry between C2A and C2B in Full-Length Synaptotagmin 1 during Ca2+-Dependent Membrane Binding

Abstract

Synaptotagmin 1 (Syt1) acts as the major calcium sensor in neuronal exocytosis. Syt1 is a synaptic vesicle-anchored membrane protein that contains two tandem Ca2+ binding C2 domains, named C2A and C2B. There is evidence that Syt1 interacts with membrane lipids and SNARE proteins simultaneously to facilitate two key steps during Ca2+-triggered membrane fusion: vesicle binding and content release. Although the overall function of Syt1 has been extensively investigated, the detailed molecular behavior of the single C2A and C2B domains in the neural regulatory process remains unclear. In particular, the differential function of the two C2 domains and how they cooperate in membrane fusion has not been determined.Here we employed EPR spectroscopy, fluorescence interference contrast (FLIC) microscopy and total internal reflection fluorescence (TIRF) microscopy to dissect the state of C2A and C2B domains in full-length Syt1 during Ca2+-dependent membrane binding. CW-EPR lineshapes and power saturation of spin-labeled positions in calcium binding loops of C2A in full-length Syt1 (1-421) and truncated Syt1 (1-266) without C2B domain suggest that C2A domain alone in Syt1 has a stronger membrane binding ability. A TIRF liposome capture assay further reveals that truncated Syt1 has a significant higher initial rate and extent in trans-binding with liposomes than full-length Syt1. Our data indicate that the C2B domain might be involved in some cis-binding thereby reducing total liposome binding. FLIC microscopy validates the strong involvement of C2A during Ca2+-dependent liposome binding.Our detailed structural and functional information thus provides a clue to differential regulatory mechanisms employed by C2A and C2B domains in full-length Syt1 interacting with Ca2+ and key membrane lipids, such as PS and PIP2.