Letter | Published:

Structural basis for maintenance of bacterial outer membrane lipid asymmetry

Abstract

The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds1 , 2. The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet1,2,3. This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane4. The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C5,6, but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

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Acknowledgements

We would like to thank the staff at beam lines I24, I04 and I04-1 of the Diamond Light Source UK for beam time (proposal mx13587) and assistance with data collection. The research of S.S.K., U.K., D.B. and B.v.d.B. has received support from the Innovative Medicines Initiatives Joint Undertaking under Grant Agreement No. 115525, resources that are composed of financial contributions from the European Union’s seventh framework programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies in-kind contribution.

Author information

B.v.d.B and J.A-R. purified MlaA proteins, crystallized MlaA–OmpF complexes, and determined crystal structures. J.A-R. constructed MlaA variant proteins and conducted in vitro functional assays. S.S.K carried out and analysed molecular dynamics simulations. U.K. supervised the computational studies. A.B. collected X-ray diffraction data and maintained the Newcastle Structural Biology Laboratory. B.C. carried out competitive fitness experiments, supervised by D.B. B.v.d.B and J.A-R designed experiments and B.v.d.B wrote the paper, with input from all co-authors.

Competing interests

The authors declare no competing financial interests.

Correspondence to Bert van den Berg.

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Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary References.

Life Sciences Reporting Summary

Supplementary Video 1

Atomistic molecular dynamics simulation (200 ns) of the KpMlaA–OmpF complex, showing the movement of the POPE molecule interacting with MlaA (oxygens, red; nitrogens, blue). MlaA is shown as a cartoon and coloured light blue (OmpF; grey). Phosphate head groups are shown as olive and green balls for the outer and inner leaflets respectively, water molecules as small blue spheres. The centre of the channel is indicated by the orange line. Residue Asp152, interacting with the POPE head group is shown.

Supplementary Video 2

Coarse-grained molecular dynamics simulation (2 μs) of the KpMlaA–OmpF complex.

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Fig. 1: MlaA is a monomeric α-helical OM protein with a central channel.
Fig. 2: MlaA binds OM phospholipids.
Fig. 3: Functional analyses of MlaA.
Fig. 4: MlaA is an outer leaflet phospholipid translocation channel.