%0 Journal Article %A Valeria Kalienkova %A Vanessa Clerico Mosina %A Laura Bryner %A Gert T. Oostergetel %A Raimund Dutzler %A Cristina Paulino %T Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM %D 2019 %R 10.1101/455287 %J bioRxiv %P 455287 %X 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 statement cryo-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. %U https://www.biorxiv.org/content/biorxiv/early/2019/02/05/455287.full.pdf