Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Characterization of roles of auxiliary proteins in an ABC transporter complex implicated in maintenance of outer membrane lipid asymmetry in Escherichia coli (#240)

Shuhua Thong 1 , Shu Sin Chng 1 2
  1. National University of Singapore, Singapore
  2. Singapore Centre on Environmental Life Sciences Engineering, National University of Singapore, Singapore, Singapore

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer consisting of lipopolysaccharides (LPS) and phospholipids (PLs) in the outer and inner leaflets, respectively. It is essential for growth, and its unique lipid asymmetry renders it an effective permeability barrier against toxic compounds including antibiotics. In Escherichia coli, the OmpC-Mla system is important for maintenance of OM lipid asymmetry, and is proposed to actively prevent PL accumulation in the outer leaflet of the OM by extracting and transporting aberrantly localized PLs back to the inner membrane (IM). This process is supported by a putative ATP-binding cassette (ABC) transporter in the IM, whose composition and function are not known. Here, we describe the biochemical characterization of this ABC transporter complex. We show that the transporter is made up of canonical components, nucleotide-binding domain MlaF and transmembrane domain MlaE, and auxiliary proteins, MlaD and MlaB. We further demonstrate that MlaD forms extremely stable hexamers within the complex and modulates ATP hydrolytic activity. In addition, we establish critical roles for MlaB in both the assembly and activity of the transporter complex. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.

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