Structural basis for phospholipid scrambling in the TMEM16 family

Abstract

Upon activation, lipid scramblases dissipate the lipid asymmetry of membranes, in an ATP-independent manner, by catalyzing flip-flop of lipids between the leaflets. The molecular identities of these proteins long remained obscure, but in recent years the TMEM16 family of proteins has been found to constitute Ca-activated scramblases. Recently, the X-ray structure of a fungal TMEM16 homologue has provided insight into the architecture of this protein family and into potential scrambling mechanisms. The protein forms homodimers with each subunit containing a membrane-spanning hydrophilic cleft. This region is of sufficient size to harbor polar headgroups on their way across the membrane and thus may lower the energetic barrier for the diffusion of lipids between the two leaflets of the bilayer. A regulatory Ca binding site located within the membrane adjacent to this hydrophobic cleft is responsible for activation by yet unknown mechanisms.

Lipids and Ions Traverse the Membrane by the Same Physical Pathway in the nhTMEM16 Scramblase

“…proteins reconstituted in liposomes that show substantial scramblase activity in the absence of Ca 2+ (Malvezzi et al, 2013;Brunner et al, 2016). Less than 20% of untransfected cells scramble under similar conditions, as we reported previously (Yu et al, 2015).…”

“…The X-ray crystallographic structure of fungal nhTMEM16 (4WIT) at 3.4 Å resolution (Brunner et al, 2016) was used as the starting structure for simulation. Missing loops (residues 1-18, 130-140, 465-482, 586-593, 657-659, 685-691 and 720-735) were added using SuperLooper (Hildebrand et al, 2009) which models loops based on templates from protein structures to fill in gaps in the protein structure.…”

“…Phospholipid scrambling is mediated by phospholipid scramblases, which harvest the energy of the phospholipid gradient to drive the nonspecific and bidirectional transport of phospholipids between leaflets. Proteins responsible for Ca 2+ -activated lipid scrambling belong to the anoctamin/TMEM16 superfamily (Suzuki et al, 2010(Suzuki et al, , 2013Pedemonte and Galietta, 2014;Picollo et al, 2015;Brunner et al, 2016;Whitlock and Hartzell, 2017). Among the ten mammalian TMEM16 paralogs, TMEM16C, D, F, G, and J have been identified as Ca 2+ -activated phospholipid scramblases while TMEM16A and TMEM16B are Ca 2+ -activated Cl À channels (CaCCs).…”

“…The scramblases and ion channels are thought to share a common architecture and mode of Ca 2+ activation, but the conduction pathways for ion and lipid transport remain incompletely understood. The fungal nhTMEM16 (Brunner et al, 2014(Brunner et al, , 2016 was recently crystalized and has become an important model for structural and functional investigation of TMEM16s. nhTMEM16 is a homodimer with 10 transmembrane helices in each subunit.…”

Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity

“…By FRET, we found evidence that the last part of the C-terminus of one TMEM16A monomer is indeed close to the N-terminus of the other monomer. Our results are therefore in agreement with the structure postulated for nhTMEM1612, a distant fungal paralog of TMEM16A, suggesting a common overall spatial organization of TMEM16 proteins working as channels and scramblases19. By intermolecular cross-linking, we found results consistent with a close distance between the most proximal part of each C-terminus and the first intracellular loop of the opposite subunit.…”