Electrostatics and Local Aromatic Residues Govern Lipid Binding and Membrane Penetration of Synaptotagmin C2 Domains

<jats:title>Abstract</jats:title> <jats:p>Synaptotagmins (Syts) are Ca²⁺-sensing exocytosis regulators whose tandem C2 domains interact with phosphoinositides and membranes to trigger neurotransmitter and hormone release. Although Ca²⁺ binding is known to enhance C2 domain-membrane interactions, the sequence determinants governing lipid binding and membrane penetration across Syt isoforms remain incompletely understood.</jats:p> <jats:p>Here, we performed MARTINI coarse-grained molecular dynamics simulations of isolated C2A and C2B domains from eight Ca²⁺-sensing Syt isoforms (Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9, and Syt10) interacting with phosphatidylinositol 4,5-bisphosphate (PIP₂)-containing plasma membranes. To systematically modulate electrostatic properties, we introduced partial and full charge-flip mutations at conserved acidic residues within the calcium-binding loops (CBLs). By integrating simulations across multiple isoforms and charge states, we sought to identify the dominant sequence determinants governing membrane interactions.</jats:p> <jats:p>We found that PIP₂ binding to both, CBLs and polybasic patches (PBs), is associated with loop net charge, yielding correlations &gt; 0.95 across all isoforms. However, membrane penetration is not sufficiently explained by loop net charge alone. The local phenylalanines additionally increase membrane penetration independent of loop net charge.</jats:p> <jats:p>Together, these findings establish a comprehensive electrostatic–aromatic framework where loop net charge governs PIP₂ binding, whereas loop net charge and local phenylalanine enrichment jointly govern membrane penetration across Syt C2 domains.</jats:p>