X-ray structure of a calcium-activated TMEM16 lipid scramblase

Abstract

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.

Atomistic insight into lipid translocation by a TMEM16 scramblase

“…Further details on the continuum calculations can be found in SI Materials and Methods. All MD simulations used the 4WIS structure of nhTMEM16 (7). Missing loops were built with MODELLER 9.15 (34).…”

“…1A) (7). The protein forms a dimer, and each subunit has a hydrophilic groove composed of polar and charged residues that face the membrane core and traverses the entire bilayer (blue strip in Fig.…”

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

“…Residues studied in the present work are inside of-or in close proximity to-the so-called subunit cavity, determined by a narrow crevice that spans the entire membrane. 5 In addition, according to findings by Qu and Hartzell, it may be suggested that TMEM16A CaCC have a coneshaped pore, with the largest opening facing the extracellular medium, and with blockers entering the pore from the extracellular side. 10 Two anion-binding sites with electrical distances of 0.3 and 0.5-0.6 are likely to be present in the ion permeation pathway.…”

“…3,4 Although the crystal structure of a TMEM16 protein has been reported, the structure of the pore of these channels is still enigmatic. 5 In this context, we continued our study of residues which could be important for the determination of the properties of the pore of Xenopus tropicalis TMEM16A (xtTMEM16A). We have previously reported 6 that positively charged residues R646 and R761 are important for ion permeation.…”

“…In fact, the crystal structure indicates that TM5 forms part of the protein surface inside the subunit cavity, which includes residues previously related to channel function. 5 Thus, we worked with mutants R646E, R761E, the double mutant (R646ECR761E) and the newly generated mutants in TM5 (namely, K613E, K628E and K630E), and we characterized the permeation properties of these mutants. In addition, we studied whether the mutations affected blocker interactions within the pore, based on the assumption that the blockers may act as organic anions, which might interact-much like inorganic anions do-with positively charged residues in the permeation pathway of the pore.…”