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 between SYT1/2 and BoNT/B1, these structures neglect the membrane environment, potentially overlooking critical interactions. Here, we used molecular dynamics simulations of membrane-inserted SYT1 and SYT2 in lipid rafts to uncover how membrane constraints reshape their juxtamembrane domains and enable unique contacts with the BoNT/B1’s lipid-binding loop (LBL). These interactions, absent in soluble crystal structures, reconcile experimental findings from nanodisc and mutagenesis assays. Notably, in the SYT1 complex, ganglioside GT1b dynamically bridges the gap created by membrane stress, stabilizing the SYT1-BoNT/B1 interface while SYT2 remains stably engaged. Our results demonstrate that lipid rafts and intrinsically disordered protein domains act synergistically to create functional binding modes missed by crystallography or AI-based structure prediction. This study highlights the epigenetic dimension of protein structure (EDPS), where environmental constraints dictate receptor conformation, and provides mechanistic insights for designing BoNT/B1 antidotes or therapeutics that exploit lipid-assisted binding.