Structural Basis of Lipid Scrambling and Ion Conduction by TMEM16 Scramblases

Abstract

The plasma membrane of eukaryotic cells is asymmetric, with polar and charged lipids sequestered to the inner leaflet when the cell is at rest. Activation of phospholipid scramblases rapidly collapses this asymmetry and externalizes negatively charged phosphatidylserine lipids, activating extracellular signaling networks that control processes such as apoptosis, blood coagulation, membrane fusion and repair. The TMEM16 family is comprised of Ca2+‐dependent Cl− channels and phospholipid scramblases with non‐selective ion channel activity. Structural and functional analyses of TMEM16 scramblases identified a membrane‐exposed hydrophilic groove that serves as a dual translocation pathway for ions and lipids. The mechanisms underlying Ca2+‐dependent gating of TMEM16 scramblases/non‐selective channels, lipid permeation and the emergence of their ion conduction pore remain poorly understood. We used cryo‐electron microscopy to determine the structures of a fungal scramblase/non‐selective channel from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs in the presence and absence of Ca2+. These structures reveal that Ca2+ binding induces a global rearrangement of the transmembrane and cytosolic regions, resulting in opening of the lipid permeation pathway by way of rearrangements of TM4 and TM6. The structures of the scramblase/nanodisc complex in the presence and absence of Ca2+ show that the scramblases induce a pronounced distortion of the surrounding membrane: the outer leaflet is bent along the dimer axis of the protein and the membrane is thinned near the opened lipid translocation pathway thereby facilitating transbilayer lipid movement.Molecular dynamics simulations of a second closely related fungal TMEM16 scramblase, nhTMEM16, revealed that a hydrophobic lock between TM3 and TM4 is essential to maintain the open, lipid‐conductive conformation of the scramblase. MD simulations suggest that disruption of this lock favors the positioning of TM4 giving rise to a Ca2+‐bound conformation that is closed to the membrane and thus non‐conductive for lipids. Indeed, mutations at the TM3/TM4 lock severely impair lipid scrambling. Notably, these mutants retain ion channel activity, indicating that the dual activity scramblase/channel was converted into a channel only protein. The cryoEM structure of a channel‐only mutant reveal a continuous, protein‐delimited pore of sufficient size to allow ion permeation. Our results show that TM6 and TM4 are the gating elements of TMEM16 scramblases where TM6 movement is directly controlled by Ca2+‐binding and TM4 rearrangements are controlled by a hydrophobic lock between TM3 and TM4.Support or Funding InformationNIGMS 5R01 GM106717