Structure of bacterial phospholipid transporter MlaFEDB with substrate bound.

Nicolas Coudray,Mariyah N Saiduddin,Damian C Ekiert,Georgia L Isom,Gira Bhabha,Mark R MacRae

doi:10.7554/elife.62518

Abstract

In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.

Highlights

The bacterial outer membrane (OM) is a critical barrier that protects the cell from antibiotics and other environmental threats, and protects pathogenic bacteria from the anti-microbial responses of the host
We resolved both coils of the membrane scaffold protein (MSP) belt surrounding the nanodisc (Bayburt et al, 2002) using the map filtered at 6 A, thereby clearly defining the position of the transmembrane domain (Figure 1E, Figure 1—figure supplement 3A,B)
This MCE ring is anchored in place by six MlaD transmembrane helices, which dock around the periphery of the MlaE transmembrane domains (TMDs) (Figure 1F)

Summary

Introduction

The bacterial outer membrane (OM) is a critical barrier that protects the cell from antibiotics and other environmental threats, and protects pathogenic bacteria from the anti-microbial responses of the host. Mla trafficks phospholipids between the IM and OM and is important for maintaining the outer membrane barrier (Malinverni and Silhavy, 2009; Chong et al, 2015; Thong et al, 2016; Abellon-Ruiz et al, 2017; Ekiert et al, 2017; Isom et al, 2017; Shrivastava et al, 2017; Powers and Trent, 2018; Yeow et al, 2018; Ercan et al, 2019; Hughes et al, 2019; Kamischke et al, 2019; Shrivastava and Chng, 2019) This system consists of three main parts: (1) an IM ABC transporter complex, MlaFEDB; (2) an OM complex, MlaA-OmpC/F; and (3) a soluble periplasmic protein, MlaC, which has been proposed to shuttle phospholipids between MlaFEDB and MlaA-OmpC/F (Figure 1A). A structure of the MlaFEDB complex may provide important insights into the mechanisms of bacterial lipid transport, as well as the evolution and function of the MlaE/YrbE transporters, which are conserved from double-membraned bacteria to chloroplasts

Results

E Chromosome WT

A Side view periplasm

B WT Tyr81 Val77 Leu78 Leu70 Leu99 Trp149 Phe209

Discussion

Materials and methods