RT Journal Article SR Electronic T1 Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM JF bioRxiv FD Cold Spring Harbor Laboratory SP 455287 DO 10.1101/455287 A1 Valeria Kalienkova A1 Vanessa Clerico Mosina A1 Laura Bryner A1 Gert T. Oostergetel A1 Raimund Dutzler A1 Cristina Paulino YR 2019 UL http://biorxiv.org/content/early/2019/02/05/455287.abstract AB 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.