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 murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca2+ define the ligand-free closed conformation of the protein and the structure of a Ca2+-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.

The exposure of the negatively charged lipid phosphatidylserine on the cell-surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The protein XKR9 is a member of a conserved family that has been associated with apoptotic lipid scrambling. Here, we describe structures of full-length and caspase-treated XKR9 from Rattus norvegicus in complex with a synthetic nanobody determined by cryo-electron microscopy. The 43 kDa monomeric membrane protein can be divided into two structurally related repeats, each containing four membrane-spanning segments and a helix that is partly inserted into the lipid bilayer. In the full-length protein, the C-terminus interacts with a hydrophobic pocket located at the intracellular side acting as an inhibitor of protein function. Cleavage by caspase-3 at a specific site releases 16 residues of the C-terminus thus making the pocket accessible to the cytoplasm. Collectively, the work has revealed the unknown architecture of the XKR family and has provided initial insight into its activation by caspases.

TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca2+. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.

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 Ca 2+ -bound and Ca 2+ -free states display a striking similarity to the scramblingincompetent 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. Keywords:TMEM16 family, lipid scramblase, membrane protein structure, transport mechanism, cryo-EM, single particle of the family, TMEM16F appears, with respect to phylogenetic relationships, as intermediate between the two proteins. Although working as lipid scramblase (Suzuki et al., 2010; Watanabe et al., 2018), it is closer related to the ion channel TMEM16A than to nhTMEM16 ( Figure S1A).Moreover, whereas scrambling-related ion conduction was found to be a feature of several family members (Lee et al., 2016;Malvezzi et al., 2018; Whitlock and Hartzell, 2016b), TMEM16F is the only lipid scramblase for which instantaneous calcium-activated currents were recorded in excised patches (Yang et al., 2012). To better understand how the small sequence differences in TMEM16F give rise to its distinct functional properties, we determined its structure by cryo-EM in Ca 2+ -bound and Ca 2+ -free states, both in a detergent and in a lipid environment. In parallel, we characterized the lipid transport properties of TMEM16F in vitro after reconstitution of the protein into liposomes, as well as ion conduction in transfected cells by electrophysiology. Collectively, our study reveals the architecture of TMEM16F, defines conformational changes upon ligand binding and suggests potential mechanisms for ion and lipid movement. In the most plausible scenario, both transport processes, although activated by the same mechanism, are mediated by distinct protein conformations which are at equilibrium in a calcium-bound state. RESULTS Functional properties of TMEM16FIn our study, we explored the relationship of the TMEM16F structure to its diverse functional properties. For this purpose, we have expressed TMEM16F in HEK293 cells and purified it in the detergent digitonin ( Figures S1B and S1C). To confirm that the purified protein has retained its activity as a lipid scramblase, we have investigated lipid transport with proteoliposomes using an assay that was previously established for fungal TMEM16 scramblases (Malvezzi et al., 2013;Ploier and Menon, 2016). Our data reveals a Ca 2+ -induced ...

The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the SLC4 family. A novel conserved Zn2+-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.