Robust lipid scrambling by TMEM16 proteins requires an open groove and thin membrane

Biological membranes are complex and dynamic structures with different populations of lipids on their inner and outer leaflets. The Ca<2+/>-activated TMEM16 family of membrane proteins play an important role in collapsing this asymmetric lipid distribution by spontaneously, and bidirectionally, scrambling phospholipids between the two leaflets, which can initiate signaling and alter the physical properties of the membrane. While evidence shows that lipid scrambling occurs via an open hydrophilic pathway ("groove") that spans the membrane, it remains unclear if all family members facilitate lipid movement in this manner. Here we present a comprehensive computational study of lipid scrambling by all TMEM16 members with experimentally solved structures. We performed coarse-grained molecular dynamics (MD) simulations of 27 structures from five different family members solved under activating and non-activating conditions, and we captured over 700 scrambling events in aggregate. This enabled us to directly compare scrambling rates, mechanisms, and protein-lipid interactions for fungal and mammalian TMEM16s, in both open (Ca<2+/>-bound) and closed (Ca<2+/>-free) conformations with statistical rigor. We show that nearly all (>90%) scrambling occurs in the dilated groove and the degree of induced membrane thinning positively correlates with the scrambling rate. Surprisingly, we also observed 60 scrambling events that occurred outside the canonical groove, over 90% of which took place at the dimer-dimer interface in mammalian TMEM16s. This new site suggests an alternative mechanism for lipid scrambling in the absence of an open groove