Paulino, C. | bioRxiv Science

bioRxiv2018

Cryo-EM structures and functional characterization of the lipid scramblase TMEM16F

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of TMEM16F that underlie its function as lipid scramblase and ion channel. The cryo-EM structures of TMEM16F in Ca2+-bound and Ca2+-free states display a striking similarity to the scrambling-incompetent anion channel TMEM16A, yet with distinct differences in the catalytic site and in the conformational changes upon activation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, which likely resembles an equivalent process defined in the homologue nhTMEM16, both functions appear to be mediated by alternate protein conformations, which are at equilibrium in the ligand-bound state.

biochemistry↗

bioRxiv2018

Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+-binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement. Impact statementcryo-EM reveals the properties of distinct conformations occupied during activation of the lipid scramblase nhTMEM16 and provides new insights into its interactions with the lipid environment.

biochemistry↗