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.
Integral membrane proteins fulfil important roles in many crucial biological processes, including cell signalling, molecular transport and bioenergetic processes. Advancements in experimental techniques are revealing high resolution structures for an increasing number of membrane proteins. Yet, these structures are rarely resolved in complex with membrane lipids. In 2015, the MemProtMD pipeline was developed to allow the automated lipid bilayer assembly around new membrane protein structures, released from the Protein Data Bank (PDB). To make these data available to the scientific community, a web database (http://memprotmd.bioch.ox.ac.uk) has been developed. Simulations and the results of subsequent analysis can be viewed using a web browser, including interactive 3D visualizations of the assembled bilayer and 2D visualizations of lipid contact data and membrane protein topology. In addition, ensemble analyses are performed to detail conserved lipid interaction information across proteins, families and for the entire database of 3506 PDB entries. Proteins may be searched using keywords, PDB or Uniprot identifier, or browsed using classification systems, such as Pfam, Gene Ontology annotation, mpstruc or the Transporter Classification Database. All files required to run further molecular simulations of proteins in the database are provided.
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 the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific 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 calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending 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. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy. 4 Cells and their organelles are enclosed by lipid bilayers and the lipid composition of either side of these membranes is controlled by active transporters (flippases and floppases) and passive scramblases, which equilibrate lipids between the membrane leaflets 1 . Many lipids are synthesized on the cytoplasmic side of the endoplasmic reticulum (ER) membrane which, unlike the plasma membrane (PM), has a symmetrical lipid distribution, suggesting a role for scramblases in the ER. To date, specific ER scramblases have not been identified and characterised. The ten members of the TMEM16 scramblase/channel family of integral membrane proteins show a surprising diversity of function, being either Ca 2+ -activated chloride channels (TMEM16A and B) 2-4 , or Ca 2+ -activated lipid scramblases with nonselective ion channel activity (TMEM16C, D, F, G and J) 5-8 . While some members of the family (A,B,F)reside in the plasma membrane, others, including TMEM16K 9 may function in intracellular membranes. TMEM16K is a widely distributed 10 , but relatively unstudied member of the TMEM16K family. Truncations and missense variants of TMEM16K (ANO10) are associated with the autosomal recessive spinocerebellar ataxia SCAR10 11,12 (as known as ARCA3 13-15 or ATX-ANO10 16 ). SCAR10 causing cerebellar ataxia, epilepsy and cognitive impairment with cerebellar atrophy noted on MRI brain and coenzyme Q10 deficiency found in muscle biopsy, fibroblasts and cerebrospinal fluid 11,12,17,18 . Some patients also have epilepsy and cognitive impairment 13,14 . Knockout studies in Drosophila 19 and mice 20 have suggested that loss of TMEM16K homologue function affects spindle formation 19 , Ca 2+ signalling 20 and apoptosis 19,20 .Structural studies have gone some way towards explaining how TMEM16 family members function as channels or lipid scrambla...
SignificanceOuter-membrane porins are often considered as passive conduits of small molecules across lipid bilayers. Using native mass spectrometry experiments we identify a pH-sensitive lipid-binding mechanism of outer membrane porin F, which enables increased threading of a colicin-derived peptide through open channels. Supported by molecular dynamics simulations and channel recording experiments, we posit that this mechanism attenuates channel opening in response to changes in environmental conditions, specifically pH. These findings have important consequences for mass spectrometry experiments, wherein the role of charge is often overlooked, and they also could help provide understanding of antibiotics that gain access to Gram-negative bacteria through porin-mediated pathways.
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