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

Abstract

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.

Identification and characterisation of a phospholipid scramblase in the malaria parasite Plasmodium falciparum

“…These experimental conditions resulted in an approx. 80% decrease of initial NBD-fluorescence recorded (50% quenching of fluorophores in the outer leaflet upon sodium dithionate treatment and a further 30% decrease due to translocase activity), which is well in line with established maximal activities of 80-85% in other proteins with PL scramblase function(46)(47)(48). The reported PL translocation rates for hPLSCR1 appear to be lower, most likely due to the use of NBD-PC as a substrate (with rates similar to those measured for NBD-PS in PfPLSCR reconstituted vesicles) and/or insufficient reconstitution of recombinant hPLSCR1 into liposomes(20,(23)(24)(25).…”

Membrane lipids are both the substrates and a mechanistically responsive environment of TMEM16 scramblase proteins

“…Lipids could be envisioned to slide through the polar groove by populating it with their hydrophilic headgroups while keeping the hydrophobic tails in the bilayer environment. Together, several breakthrough structures of fungal TMEM16 PLS brought evidence for global rearrangements induced by Ca 2+ ‐binding that result in the opening of a membrane‐exposed pathway through which lipid headgroups can move between leaflets . This mechanism of lipid scrambling is consistent with the proposed “credit card‐reader” model and indeed has been observed in several molecular dynamics (MD) simulations of Ca 2+ ‐bound nhTMEM16 protein in which lipids flip between the two leaflets of the membrane by engaging with various sites along the groove with their head‐groups.…”

“…In this occluded conformation, the middle region of TM4 is repositioned closer to TM6 (Fig. S3B) and the state resembles the intermediate state described in the recent cryo‐electron microscopy (cryo‐EM) studies of the nhTMEM16 and TMEM16F scramblases in lipid nanodiscs . The mechanistic underpinnings and functional consequences of this intermediate state are described elsewhere (Ref.…”

“…The deformation of the membrane surrounding the inserted TMEM16 observed both in the simulations and in the determined structures, led to the suggestion that the membrane environment plays a regulatory role in the functional dynamics of TMEM16 PLS, as discussed below. As the recent advances in structure determination of TMEM16 PLS in lipid nanodiscs have brought to light important structural details regarding the organization of the nanodisc membrane around the scramblase proteins, they offer invaluable insights regarding the active participation of the lipid environment in enabling the different functional states of these proteins.…”

“…The studies on afTMEM16 were done using relatively small size (~120 Å in diameter) nanodiscs composed of 3:1 mixture of POPE:POPG lipids and surrounded by the MSP1E3 scaffold protein. The work on nhTMEM16 and TMEM16F scramblases used larger (~185 Å in diameter) nanodiscs stabilized by a different scaffold protein, MSP2N2, and composed of either 7:3 or 3:1 mixtures of POPC:POPG lipids.…”