Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum

Alice Verchère,Michael A Goren,Wei-Lin Ou,Anant K Menon,George Khelashvili,Birgit Ploier,Peter Bütikofer,Takefumi Morizumi,Oliver P Ernst

doi:10.1038/s41598-017-09835-5

Abstract

The retinylidene protein bacteriorhodopsin (BR) is a heptahelical light-dependent proton pump found in the purple membrane of the archaeon Halobacterium salinarum. We now show that when reconstituted into large unilamellar vesicles, purified BR trimers exhibit light-independent lipid scramblase activity, thereby facilitating transbilayer exchange of phospholipids between the leaflets of the vesicle membrane at a rate >10,000 per trimer per second. This activity is comparable to that of recently described scramblases including bovine rhodopsin and fungal TMEM16 proteins. Specificity tests reveal that BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggest that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, extensive coarse-grained molecular dynamics simulations of a BR trimer in an explicit phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These results suggest that the lipid translocation pathway may lie at or near the interface of the protomers of a BR trimer.

Highlights

Phospholipids move rapidly within biological membranes, with in-plane rotational and lateral diffusion occurring on a nanosecond time scale[1]
We found that the number of penetrant water molecules depended on the Y147 residue from transmembrane helix 5 of the adjacent protomer suggesting that the lipid translocation pathway may go through the interface of the protomers of a BR trimer
Size exclusion chromatography (SEC) revealed a single monodisperse peak (Fig. 1b) that eluted at a position consistent with trimeric BR in a dodecyl β-D-maltoside (DDM) micelle[23]

Summary

Introduction

Phospholipids move rapidly within biological membranes, with in-plane rotational and lateral diffusion occurring on a nanosecond time scale[1]. Scramblase proteins eluded molecular identification until recently when two scramblases were conclusively identified and their transport activities verified by biochemical reconstitution of purified proteins into large unilamellar vesicles[1]. Both scramblases are members of large protein families - Class A G protein-coupled receptors exemplified by the visual pigment rhodopsin[8], and www.nature.com/scientificreports/. Consistent with this proposal, extensive coarse-grained molecular dynamics simulations of a BR trimer in an explicit phospholipid membrane revealed a number of water molecules along transmembrane helix 1, demonstrating the polarity of the proposed pathway. We found that the number of penetrant water molecules depended on the Y147 residue from transmembrane helix 5 of the adjacent protomer suggesting that the lipid translocation pathway may go through the interface of the protomers of a BR trimer

Methods

Results

Conclusion