Functional Characterization of Calcium-activated Phospholipid Scramblase Activity of nhTMEM16

Abstract

Asymmetric lipid distribution between the two leaflets of bilayer is a physiologically important feature of biological membranes. Dissipation of lipid asymmetry by lipid scrambling is a ubiquitous cellular mechanism essential for membrane biogenesis, cell signaling, and bacterial cell wall assembly. The recently crystallized structure of a TMEM16 family member nhTMEM16 provided the first structural insight into the mechanism of lipid scramblases, which facilitate the passive bidirectional transport of diverse lipids between the two leaflets of the membrane. It identified a conserved Ca2+-binding site harbored in a hydrophilic crevice predicted to face the hydrophobic core of the membrane and involve in lipid scrambling, although no lipids were resolved in the structure. In order to investigate the Ca2+-mediated scrambling mechanism, identify the lipid transport pathway, and characterize the role of Ca2+, we have performed extended equilibrium molecular dynamics (MD) simulations with nhTMEM16 embedded in various lipid bilayers (POPC, POPE, POPS, and mixtures thereof) in the presence or absence of Ca2+. Our simulations reveal a previously unidentified spiral membrane-traversing hydrophilic track around the protein surface, connecting the inner and outer leaflets. Especially in the Ca2+-bound systems, lipids from the inner and outer leaflets spontaneously bind to the hydrophilic track with their headgroups and propagate toward the center of the membrane. In the Ca2+-free systems, on the other hand, the hydrophilic track is observed to substantially narrow allowing only smaller species such as water and ions to bind. Furthermore, umbrella sampling (US) simulations are used to characterize the complete lipid transport pathway and to identify key residues and energetics of the lipid translocation. Our study provides crucial atomic details of the scrambling pathway for the first time, and uncovers the nature of Ca2+-dependence by revealing the structural rearrangements of the hydrophilic track for lipid translocation.