Role of Hydrophilic Groove of the TMEM16 Protein in Lipid Scrambling

Abstract

The TMEM16 proteins are Ca2+-dependent ion channels and/or phospholipid scramblases that play key roles in a variety of physiological functions. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca2+-dependent externalization of phosphatidylserine (PS) in activated platelets, suggesting that this homologue functions as a scramblase. Explicit demonstration that some TMEM16 proteins are Ca2+-dependent phospholipid scramblases comes from reconstitution and assay of purified fungal TMEM16 homologs (afTMEM16 and nhTMEM16). The recently determined crystal structure of nhTMEM16, revealed a dimeric arrangement of the protein and the presence of hydrophilic groove on the opposite sides of the dimer interface exposed to the lipid membrane. Extensive mutagenesis studies have established the importance of the groove in the lipid scrambling by showing that mutations of specific residues in the groove region to either Trp or Ala significantly reduce the kinetics of lipid translocation. We have used a combination of unbiased and enhanced sampling atomistic molecular dynamics (MD) simulations of nhTMEM16 in lipid membrane and water environments to evaluate the structural context and detailed mechanisms for the measured changes in scrambling efficiency by mutant nhTMEM16 constructs. The findings center on the formation and mechanistic role of a continuous water pathway along the membrane-facing surface of the groove in the wild type protein that enables lipid translocation. The manner in which the mutations cause a significant reduction in the level of the hydration in the groove region points to a key role of the hydrophilic groove of TMEM16 in lipid scrambling.