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.

11Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-12 ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca 2+ -13 dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used 14 cryo-electron microscopy and functional assays to address this question. Ca 2+ -bound and Ca 2+ -free 15 conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its 16 environment and they reveal the conformational changes underlying its activation. In this process, 17 Ca 2+ -binding induces a stepwise transition of the catalytic subunit cavity, converting a closed 18 cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that 19 becomes accessible to lipid headgroups in the Ca 2+ -bound state. Additionally, our structures 20 demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, 21 thereby decreasing the energy barrier for lipid movement. 22 23 24 Impact statement: cryo-EM reveals the properties of distinct conformations occupied during 25 activation of the lipid scramblase nhTMEM16 and provides new insights into its interactions with 26 the lipid environment. 27 65 furrow for the permeation of lipids upon Ca 2+ binding in a stepwise process involving coupled 66 ligand-dependent and ligand-independent steps. 67 68Results 69 Structural characterization of nhTMEM16 in detergent 70For our investigations of the ligand-induced activation mechanism of the lipid scramblase 71 nhTMEM16, we have recombinantly expressed the protein in the yeast S. cerevisiae, purified it in 72 the detergent n-Dodecyl β-D-maltoside (DDM) in the absence of Ca 2+ and added Ca 2+ during 73 5 sample preparation for structure determination and transport experiments. We have reconstituted 74 the purified protein into liposomes of different composition containing fluorescently labeled lipids 75 and characterized lipid transport using an assay that follows the irreversible decay of the 76 fluorescence upon addition of the membrane-impermeable reducing agent dithionite to the outside 77 of the liposomes (Brunner et al., 2014; Malvezzi et al., 2013; Ploier and Menon, 2016). In these 78 experiments we find a Ca 2+ -dependent increase of transport but also a pronounced basal activity 79 of the protein in absence of Ca 2+ , which was previously described as a hallmark of fungal 80 TMEM16 scramblases (Brunner et al., 2014; Malvezzi et al., 2013) (Figure 1-figure supplement 81 1). For the structural characterization of nhTMEM16, we have initially studied Ca 2+ -bound and 82 Ca 2+ -free samples in detergent by cryo-EM. By this approach, we have obtained two datasets of 83 high quality and of sufficient resolution (i.e. 3.6 Å for Ca 2+ -bound and 3.7 Å for Ca 2+ -free 84 conditions) for a detailed interpretation by an atomic model (Figure 1; Figure 1-figure supplements 85 2-4; Table 1). In the structure determined in the presence of Ca 2+ , we find a conformation of t...

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