Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase.
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ABSTRACT:
SUBMITTER: Falzone ME
PROVIDER: S-EPMC6355197 | biostudies-literature | 2019 Jan
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:The transmembrane protein 16 (TMEM16) family of membrane proteins includes both lipid scramblases and ion channels involved in olfaction, nociception, and blood coagulation. The crystal structure of the fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase suggested a putative mechanism of lipid transport, whereby polar and charged lipid headgroups move through the low-dielectric environment of the membrane by traversing a hydrophilic groove on the membrane-spanning surface of the protein. Here, we use computational methods to explore the membrane-protein interactions involved in lipid scrambling. Fast, continuum membrane-bending calculations reveal a global pattern of charged and hydrophobic surface residues that bends the membrane in a large-amplitude sinusoidal wave, resulting in bilayer thinning across the hydrophilic groove. Atomic simulations uncover two lipid headgroup-interaction sites flanking the groove. The cytoplasmic site nucleates headgroup-dipole stacking interactions that form a chain of lipid molecules that penetrate into the groove. In two instances, a cytoplasmic lipid interdigitates into this chain, crosses the bilayer, and enters the extracellular leaflet, and the reverse process happens twice as well. Continuum membrane-bending analysis carried out on homology models of mammalian homologs shows that these family members also bend the membrane-even those that lack scramblase activity. Sequence alignments show that the lipid-interaction sites are conserved in many family members but less so in those with reduced scrambling ability. Our analysis provides insight into how large-scale membrane bending and protein chemistry facilitate lipid permeation in the TMEM16 family, and we hypothesize that membrane interactions also affect ion permeation.
| S-EPMC5150362 | biostudies-literature
Project description:BACKGROUND: TMEM16A and 16B work as Cl(-) channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown. RESULTS: Using TMEM16F(-/-) cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases. CONCLUSION: Some TMEM16 members are divided into two Cl(-) channels and five lipid scramblases. SIGNIFICANCE: Learning the biochemical function ofTMEM16family members is essential to understand their physiological role. Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane protein, as a Ca(2+)-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca(2+) ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F(-/-) cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca(2+)-dependent Cl(-) channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca(2+)-dependent Cl(-) channels (16A and 16B) and Ca(2+)-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).
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Project description:Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca2+ for their activation and substrate processing. However, the Ca2+-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.
| S-EPMC7206013 | biostudies-literature
Project description:Two TMEM16 family members, TMEM16A and TMEM16F, have different ion transport properties. Upon activation by intracellular Ca2+, TMEM16A-a Ca2+-activated Cl- channel-is more selective for anions than cations, whereas TMEM16F-a phospholipid scramblase-appears to transport both cations and anions. Under saturating Ca2+ conditions, the current-voltage (I-V) relationships of these two proteins also differ; the I-V curve of TMEM16A is linear, while that of TMEM16F is outwardly rectifying. We previously found that mutating a positively charged lysine residue (K584) in the ion transport pathway to glutamine converted the linear I-V curve of TMEM16A to an outwardly rectifying curve. Interestingly, the corresponding residue in the outwardly rectifying TMEM16F is also a glutamine (Q559). Here, we examine the ion transport functions of TMEM16 molecules and compare the roles of K584 of TMEM16A and Q559 of TMEM16F in controlling the rectification of their respective I-V curves. We find that rectification of TMEM16A is regulated electrostatically by the side-chain charge on the residue at position 584, whereas the charge on residue 559 in TMEM16F has little effect. Unexpectedly, mutation of Q559 to aromatic amino acid residues significantly alters outward rectification in TMEM16F. These same mutants show reduced Ca2+-induced current rundown (or desensitization) compared with wild-type TMEM16F. A mutant that removes the rundown of TMEM16F could facilitate the study of ion transport mechanisms in this phospholipid scramblase in the same way that a CLC-0 mutant in which inactivation (or closure of the slow gate) is suppressed was used in our previous studies.
| S-EPMC6445582 | biostudies-literature
Project description:Members of the TMEM16/ANO family of membrane proteins are Ca2+-activated phospholipid scramblases and/or Cl- channels. A membrane-exposed hydrophilic groove in these proteins serves as a shared translocation pathway for ions and lipids. However, the mechanism by which lipids gain access to and permeate through the groove remains poorly understood. Here, we combine quantitative scrambling assays and molecular dynamic simulations to identify the key steps regulating lipid movement through the groove. Lipid scrambling is limited by two constrictions defined by evolutionarily conserved charged and polar residues, one extracellular and the other near the membrane mid-point. The region between these constrictions is inaccessible to lipids and water molecules, suggesting that the groove is in a non-conductive conformation. A sequence of lipid-triggered reorganizations of interactions between these residues and the permeating lipids propagates from the extracellular entryway to the central constriction, allowing the groove to open and coordinate the headgroups of transiting lipids.
| S-EPMC6092359 | biostudies-literature
Project description:Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
| S-EPMC6718402 | biostudies-literature
Project description:Phospholipid (PL) scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca(2+)-gated channels have been shown to be involved in Ca(2+)-dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling. Here we show that purified afTMEM16, from Aspergillus fumigatus, is a dual-function protein: it is a Ca(2+)-gated channel, with characteristics of other TMEM16 homologues, and a Ca(2+)-dependent scramblase, with the expected properties of mammalian PL scramblases. Remarkably, we find that a single Ca(2+) site regulates separate transmembrane pathways for ions and lipids. Two other purified TMEM16-channel homologues do not mediate scrambling, suggesting that the family diverged into channels and channel/scramblases. We propose that the spatial separation of the ion and lipid pathways underlies the evolutionary divergence of the TMEM16 family, and that other homologues, such as TMEM16F, might also be dual-function channel/scramblases.
| S-EPMC3970400 | biostudies-literature
Project description:TMEM16 proteins are a recently identified protein family comprising Ca2+-activated Cl- channels that generate outwardly rectifying ionic currents in response to intracellular Ca2+ elevations. Some TMEM16 family members, such as TMEM16F/ANO6 are also essential for Ca2+-dependent phospholipid scrambling. TMEM16-like genes are present in the genomes of most eukaryotic species, the function(s) of TMEM16 family members from evolutionary ancient eukaryotes is not completely clear. Here, we provide insight into the evolution of these TMEM16 proteins by similarity searches for ancestral sequences. All eukaryotic genomes contain TMEM16 homologs, but only vertebrates have the full repertoire of ten distinct subtypes. TMEM16 homologs studied so far belong to the opisthokont branch of the phylogenetic tree, which includes the animal and fungal kingdoms. An organism outside this group is Dictyostelium discoideum, a representative of the amoebozoa group that diverged from the metazoa before fungi. We here functionally investigated the TMEM16 family member from Dictyostelium discoideum. When recombinantly expressed in HEK293 cells, DdTMEM16 induces phospholipid scrambling. However, in several electrophysiological experiments we did not find evidence for a Ca2+-activated Cl- channel function of DdTMEM16.
| S-EPMC5812556 | biostudies-literature
Project description:Activation of Ca2+-dependent TMEM16 scramblases induces the externalization of phosphatidylserine, a key molecule in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove, and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements involved in groove opening and of how lipids reorganize outside the closed groove remain unknown. Using cryogenic electron microscopy, we directly visualize how lipids associate at the closed groove of Ca2+-bound nhTMEM16 in nanodiscs. Functional experiments pinpoint the lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryoEM structure determination.
| S-EPMC10462188 | biostudies-literature