Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

Abstract

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism, revealing an inward-open conformation. Taking advantage of the genetic tractability of , we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

Loss of specificity variants of WzxC suggest that substrate recognition is coupled with transporter opening in MOP‐family flippases

“…In order to better understand the molecular basis for the effects of the MJ WzxC mutations, we constructed a homology model of WzxC using the crystal structure of E. coli MurJ (Zheng et al, ) as a template (Fig. and S5).…”

“…A homology model of E. coli WzxC was constructed in MODELLER (Webb and Sali, ) using a multi‐template modeling protocol with the crystal structures of MurJ from E. coli (Zheng et al, ) and T. africanus (Kuk and Lee, ) (PDB ID: 5T77) serving as templates.…”

“…How these transporters specifically recognize their substrates over the many different types of lipid‐linked oligosaccharides produced in a bacterial cell is not currently known. The transport mechanism is also poorly understood, but the recently solved structures of the MOP‐family protein MurJ from Thermosipho africanus and Escherichia coli has provided important clues (Kuk and Lee, ; Zheng et al, ).…”

“…Evolutionary coupling (EC) analysis (Hopf et al, ) also shows that MurJ must adopt an additional conformation not accounted for in the inward‐open crystal structure, since pairs of distant residues on the cytosolic face so strong co‐evolution. An outward‐open model of MurJ similarly leaves predicted interactions unsatisfied on the periplasmic face of the protein, indicating that at least two distinct conformations are subject to evolutionary selective pressure (Zheng et al, ). Thus, it has been proposed that MurJ and other MOPS family proteins mediate transport/flipping via an alternating access model involving the interconversion between the inward‐ and outward‐facing conformations, alternately allowing the lipid II headgroup to access the cytosolic and periplasmic faces of the membrane (Islam and Lam, ; Ruiz, ; Kuk and Lee, ; Zheng et al, ).…”

FtsW activity and lipid II synthesis are required for recruitment of MurJ to midcell during cell division in Escherichia coli

“…Depletion of MurJ will cause irregularly shaped cell, and finally result into cell lysis (Ruiz, ). Earlier functional and structural studies of MurJ reveal an outward‐facing central cavity that is formed by TMHs 1, 2, 7 and 8, which contain several charged residues that are essential for MurJ function (Butler et al, , ), while the observation of an open inward‐facing conformation in the crystal structure of MurJ from both Thermosipho africanus (MurJ TA ) and E. coli suggest alternative conformational changes of MurJ (Kuk et al, ; Zheng et al, ). In vivo evidence favors MurJ over FtsW as the lipid II flippase, and depletion or inhibition of MurJ caused the accumulation of lipid II in cells (Ruiz, ; Sham et al, ; Young, ; Qiao et al, ), and its activity is dependent on membrane potential (Rubino et al, ).…”