Structural basis for the regulation mechanism of Ca2+-dependent activity of TMEM16 scramblase.

Abstract

The collapse of the plasma membrane asymmetry by activated TMEM16 scramblases results in exposure of phosphatidylserine (PS), an essential trigger for multiple fundamental physiological processes. Functionally, Ca2+ binding to two conserved sites activates all TMEM16's scramblases. In contrast, Ca2+ binding leads to opening of a hydrophilic groove only in some homologues, whereas in others no opening is seen. Two questions thus arise: first, what are the structural determinants that enable groove opening in some TMEM16 homologues? Second, what are the conformational rearrangements that regulate Ca2+ dependent scrambling in the absence of groove opening? Here we used cryoEM to determine the structure of the fungal nhTMEM16 scramblase in nanodiscs in three states: Ca2+-free closed, Ca2+-bound closed and Ca2+-bound open. We find that the sequential appearance of two π-helical turns on the TM6 helix correlates with the stepwise conformational transitions from the Ca2+-free closed to Ca2+-bound closed and to the open state. Mutagenesis of three residues responsible for stabilizing the second π-helical turn prevented groove opening and trapped nhTMEM16 in the intermediate Ca2+-bound closed state, suggesting this is a necessary step in groove opening. The 2.6 Å resolution of the cryoEM map in the Ca2+-bound closed state enables us to visualize how lipid reorganization outside a closed groove results in a pronounced thinning of the membrane that could facilitate scrambling. In the absence of Ca2+ and in mutants displaying reduced scrambling activity we observe more pronounced bending of the TM6 and reduced thinning. We propose that the Ca2+ dependent straightening of the TM6 helix promotes scrambling by increasing membrane thinning. These findings provide the structural basis for the regulation of Ca2+-dependent activity of TMEM16 scramblase, which could be extended to other homologues with a similar fold.