Gating mechanism of the extracellular entry to the lipid pathway in a TMEM16 scramblase

Anant K Menon,Byoung-Cheol Lee,Alessio Accardi,George Khelashvili,Maria Falzone,Harel Weinstein

doi:10.1038/s41467-018-05724-1

Anant K Menon, Byoung-Cheol Lee + Show 4 more

Open Access

https://doi.org/10.1038/s41467-018-05724-1

Copy DOI

Abstract

Members of the TMEM16/ANO family of membrane proteins are Ca2+-activated phospholipid scramblases and/or Cl− channels. A membrane-exposed hydrophilic groove in these proteins serves as a shared translocation pathway for ions and lipids. However, the mechanism by which lipids gain access to and permeate through the groove remains poorly understood. Here, we combine quantitative scrambling assays and molecular dynamic simulations to identify the key steps regulating lipid movement through the groove. Lipid scrambling is limited by two constrictions defined by evolutionarily conserved charged and polar residues, one extracellular and the other near the membrane mid-point. The region between these constrictions is inaccessible to lipids and water molecules, suggesting that the groove is in a non-conductive conformation. A sequence of lipid-triggered reorganizations of interactions between these residues and the permeating lipids propagates from the extracellular entryway to the central constriction, allowing the groove to open and coordinate the headgroups of transiting lipids.

Highlights

Members of the TMEM16/ANO family of membrane proteins are Ca2+-activated phospholipid scramblases and/or Cl− channels
Two classes of ATP-driven pumps, flippases and floppases[2,3,4], create and maintain the membrane asymmetry, whose rapid collapse occurs during a process called phospholipid scrambling that externalizes PS, an effector in numerous cell activities such as blood coagulation and the phagocytic clearance of apoptotic cells[5,6,7,8,9,10]. The proteins mediating this process are called phospholipid scramblases and their identity had remained controversial until recently when members of two unrelated families of integral membrane proteins, the TMEM1611–17 and Xk-related (Xkr)[18,19,20] proteins, were identified as regulated phospholipid scramblases that respond to different physiological stimuli
While the two family-founding members, TMEM16A and B, are CaCCs25–27, it was later realized that the majority of the family members function as Ca2+-dependent phospholipid scramblases, which mediate non-selective ion transport[11,16,17,20,28]

Summary

Introduction

Members of the TMEM16/ANO family of membrane proteins are Ca2+-activated phospholipid scramblases and/or Cl− channels. Two classes of ATP-driven pumps, flippases and floppases[2,3,4], create and maintain the membrane asymmetry, whose rapid collapse occurs during a process called phospholipid scrambling that externalizes PS, an effector in numerous cell activities such as blood coagulation and the phagocytic clearance of apoptotic cells[5,6,7,8,9,10] The proteins mediating this process are called phospholipid scramblases and their identity had remained controversial until recently when members of two unrelated families of integral membrane proteins, the TMEM1611–17 and Xk-related (Xkr)[18,19,20] proteins, were identified as regulated phospholipid scramblases that respond to different physiological stimuli. Our analysis of results from molecular dynamics (MD) simulation of corresponding nhTMEM16 constructs identifies a mechanism by which lipid access to the pathway from the extracellular leaflet is controlled by dynamic rearrangements of a network of interacting polar residues whose dynamical rearrangements entail the elimination of steric constrictions along the groove, thereby enabling lipid scrambling. The extracellular region of the groove interacts with multiple lipid molecules at the extracellular region of the groove to trigger dynamic rearrangements that open the nhTMEM16 groove and allow lipid permeation

Methods

Results

Conclusion