Synaptotagmin (Syt), with at least 17 isoforms, regulates Ca 2+ -triggered exocytosis mediating neurotransmission and hormone secretion. Canonical isoforms Syt1, Syt2, Syt7, and Syt9 act through tandem C2 domains. Functionally, Syt1 and Syt2 drive synchronous neurotransmitter release, whereas Syt7 mediates asynchronous release. In pancreatic β-cells, Syt7 and Syt9 localize to distinct dense-core vesicle pools, and their loss phenocopies type 1 and 2 diabetes, respectively. The molecular dynamics underlying isoform-specific lipid binding—rapid for Syt1/2, intermediate for Syt9, and slow for Syt7—remains poorly understood. We employed MARTINI coarse-grained molecular dynamics simulations of Syt C2B domains with calcium-binding loops (CBLs) inserted into plasma membranes to probe Ca 2+ -dependent interactions. To mimic Ca 2+ -bound states, we introduced DK charge-flip mutations of two (DK2 = D303K/D365K) or five conserved residues (DK5 = D303K/D309K/D363K/D365K/D371K) in Syt1 or corresponding residues of others, mimicking multiple Ca 2+ binding of C2Bs. Simulations revealed that phosphatidylinositol (4,5)-bisphosphate (PIP2, charge -5) interacts more strongly with CBLs than phosphatidylserine (charge -1). DK2 C2Bs recruited ∼2 additional PIP2 molecules and inserted ∼2 Å deeper into membranes across all isoforms, except Syt2 and Syt7. Syt2 DK2 C2B inserted ∼3 Å deeper but bound only ∼1 additional PIP2, whereas Syt7 showed little further insertion and slower PIP2 binding. DK5 mutations of Syt2 and Syt7 bound ∼2 more PIP2, and Syt7 inserted ∼2 Å deeper than DK2, likely due to additional cationic residues (N333K in Syt2; N333R and Y364K in Syt7). In contrast, Syt1 and Syt9 showed no further increases. DR mutations of Syt1 C2B slowed PIP2 binding kinetics compared to DK without altering final occupancy. In Syt9, Syt1 residues K366 and K332 are changed to R and exhibits correspondingly slower PIP2 binding. These differences provide mechanistic insights into distinct Ca 2+ -dependent release kinetics of Syt1 vs. Syt2, and the asynchronous release mediated by Syt7.
An et al. (Sun,) studied this question.