Membrane constraints reshape synaptotagmin recognition by botulinum neurotoxin B1

Synaptotagmins 1 and 2 (SYT1 and SYT2) are essential Ca ² + sensors in neurotransmission and the functional receptors of botulinum neurotoxin B1 (BoNT/B1). While crystallographic models have defined key contacts, they neglect membrane constraints. Using molecular dynamics simulations in lipid rafts, we uncover how the membrane environment reshaped synaptotagmin conformation and enables critical contacts with BoNT/B1’s lipid-binding loop (LBL). Notably, ganglioside GT1b bridges BoNT/B1 and SYT1, stabilizing their interface through lipid-mediated interactions. This mechanism escapes AlphaFold prediction, which generates non-physiological complexes with steric clashed, revealing a fundamental limitation of current AI methods for membrane-constrained interfaces. Our study demonstrates that lipid rafts create functional binding modes through synergistic protein-lipid interactions, highlighting the epigenetic dimension of protein structure where environment dictates conformation.