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      Structural basis of Ca 2+-dependent activation and lipid transport by a TMEM16 scramblase

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          Abstract

          The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca 2+-activated scramblases, but the mechanisms underlying their Ca 2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca 2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

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          Most cited references36

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          Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.

          Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodopsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together, these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.
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            X-ray structure of a calcium-activated TMEM16 lipid scramblase.

            The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.
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              Exposure of phosphatidylserine on the cell surface.

              Phosphatidylserine (PtdSer) is a phospholipid that is abundant in eukaryotic plasma membranes. An ATP-dependent enzyme called flippase normally keeps PtdSer inside the cell, but PtdSer is exposed by the action of scramblase on the cell's surface in biological processes such as apoptosis and platelet activation. Once exposed to the cell surface, PtdSer acts as an 'eat me' signal on dead cells, and creates a scaffold for blood-clotting factors on activated platelets. The molecular identities of the flippase and scramblase that work at plasma membranes have long eluded researchers. Indeed, their identity as well as the mechanism of the PtdSer exposure to the cell surface has only recently been revealed. Here, we describe how PtdSer is exposed in apoptotic cells and in activated platelets, and discuss PtdSer exposure in other biological processes.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                16 January 2019
                2019
                : 8
                : e43229
                Affiliations
                [1 ]deptDepartment of Biochemistry Weill Cornell Medical College New YorkUnited States
                [2 ]deptDepartment of Anesthesiology Weill Cornell Medical College New YorkUnited States
                [3 ]deptDepartment of Structure and Function on Neural Network Korea Brain Research Institute DeaguRepublic of Korea
                [4 ]deptDepartment of Physiology and Biophysics Weill Cornell Medical College New YorkUnited States
                [5 ]deptDepartment of Pathology Weill Cornell Medical College New YorkUnited States
                [6 ]deptSimons Electron Microscopy Center New York Structural Biology Center New YorkUnited States
                Semmelweis University Hungary
                The University of Texas at Austin United States
                Semmelweis University Hungary
                Author information
                http://orcid.org/0000-0001-6738-7017
                http://orcid.org/0000-0002-9901-2065
                http://orcid.org/0000-0002-8014-7269
                http://orcid.org/0000-0002-6254-4447
                http://orcid.org/0000-0002-6584-0102
                Article
                43229
                10.7554/eLife.43229
                6355197
                30648972
                18f4f622-d2c5-4138-80ae-66becfbb42f6
                © 2019, Falzone et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 30 October 2018
                : 02 January 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R01GM106717
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100006984, Irma T. Hirschl Trust;
                Award Recipient :
                Funded by: Margaret and Herman Sokol Fellowship;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003725, National Research Foundation of Korea;
                Award ID: 2013R1A6A3A03064407
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100009344, Agouron Institute;
                Award ID: F00316
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000893, Simons Foundation;
                Award ID: 349247
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: GM103310
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: 1R01GM124451-02
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003621, Ministry of Science, ICT and Future Planning;
                Award ID: 18-BR-01-02
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;
                Award ID: R21NS10451
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Structural Biology and Molecular Biophysics
                Custom metadata
                Structures of a TMEM16 phospholipid scramblase reveal that its Ca 2+-dependent activation entails global conformational changes and how these rearrangements affect the membrane to enable transbilayer lipid transfer.

                Life sciences
                scrambling,membrane structure,membrane channels,phospholipids,s. cerevisiae
                Life sciences
                scrambling, membrane structure, membrane channels, phospholipids, s. cerevisiae

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