Abstract
The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology. Bacteria use multiprotein machineries for the synthesis of the PG cell wall during cell division and elongation that can be targeted by antibiotics such as the β-lactams. Lipid II, the lipid-linked precursor for PG biogenesis, is synthesized in the inner leaflet of the cytoplasmic membrane and then translocated across the bilayer, where it is ultimately polymerized into PG. In Escherichia coli, MurJ, a member of the MOP exporter superfamily, has been recently shown to have lipid II flippase activity that depends on membrane potential. Because of its essentiality, MurJ could potentially be targeted by much needed novel antibiotics. Recent structural information suggests that a central cavity in MurJ alternates between inward- and outward-open conformations to flip lipid II, but how these conformational changes occur are unknown. Here, we utilized structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe the conformational changes of MurJ in E. coli cells. We found that paired cysteine substitutions in transmembrane domains 2 and 8 and periplasmic loops of MurJ could be cross-linked with homobifunctional cysteine cross-linkers, indicating that MurJ can adopt both inward- and outward-facing conformations in vivo Furthermore, we show that dissipating the membrane potential with an ionophore decreases the prevalence of the inward-facing, but not the outward-facing state. Our study provides in vivo evidence that MurJ uses an alternating-access mechanism during the lipid II transport cycle.
Highlights
The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology
3 The abbreviations used are: IM, inner membrane; PG, peptidoglycan; glycan strands of alternating N-acetylglucosamine (GlcNAc), N-acetylglucosamine; MurNAc; N-acetylmuramic acid; Und-P, undecaprenyl phosphate; lipid I, Und-PP-MurNAc-pentapeptide; lipid II, Und-PP-MurNAc-pentapeptide-GlcNAc; MOP, multidrug/oligosaccharidyl lipid/polysaccharide; SCAM, substituted-cysteine accessibility method; two membranes, there is an aqueous compartment, known as the periplasm, that houses an essential cell wall made of peptidoglycan (PG) [1]
We identified 11 pairs of residues in the cytoplasmic side of MurJ whose C␣ positions allowed us to predict that cysteine replacements would be too far to be cross-linked in the inward-open state, yet would be within cross-linking distance in the outward-open state (Fig. 1 and Table 1)
Summary
The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology. It was predicted that TMs 1, 2, 7, and 8 of MurJ form a central hydrophilic cavity, and, in agreement with its role as a lipid II flippase, several charged residues in this cavity were shown to be required for MurJ function [26, 28] This suggested that MurJ might function to MATE exporters, which utilize an antiport mechanism that relies on a gradient of either cations or protons across the membrane to translocate toxic drugs from the cytoplasm to the periplasm [14, 17]. We propose that MurJ functions by a cationantiport, alternating-access mechanism to translocate lipid II across the inner membrane
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