X-Ray Structure of a Calcium Activated TMEM16 Lipid Scramblase

Abstract

The TMEM16s or anoctamins constitute a class of eukaryotic membrane proteins that in mammals contain ten members with high sequence conservation. Despite their close relationship these proteins are characterized by a remarkable functional diversity. The family includes the long sought-after Ca2+-activated chloride channels (TMEM16A and B) but also cation channels and lipid scramblases that support the exchange of lipids between the inner and outer leaflets of the bilayer in an ATP-independent manner. As part of the blood coagulation process, TMEM16F triggers the exposure of phosphatidylserine in blood platelets upon activation by Ca2+. TMEM16C, D, G and J were suggested to work as scramblases as well, but with variable characteristics. Although we have by now gained considerable insight into the functional properties of certain family members, their architecture and the relation to mechanisms of action were so far unknown. Here we present the crystal structure of nhTMEM16, a fungal family member that operates as a Ca2+-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that resembles a ‘spiral staircase’ and is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbors a conserved Ca2+-binding site located within the hydrophobic core of the membrane. Ca2+ binding by six residues, five of which carry a negative charge, controls the activation of scrambling in nhTMEM16. A triple mutant of residues in this site shows only weak scrambling activity that is no longer enhanced by Ca2+.The structure thus reveals the general architecture of the family and its mode of Ca2+-activation. It provides insights into potential scrambling mechanisms and will furthermore serve as a framework to unravel the conduction of ions in certain TMEM16 proteins.