Detection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling

Abstract

Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.

High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae

“…Assuming MurJ and CpsJ function similarly, Cps10BJ is expected to transition between the inward-open and outward-open states during substrate translocation. If so, mutations that promote the conformational change upon substrate binding may allow CpsJ to translocate a noncognate substrate ( 40 ). Based on this assumption, residue F109 in Cps10BJ lies in the putative extracellular gate (see Fig.…”

“…This step is thought to be mediated by the widely conserved MOP-family flippase CpsJ ( 22 ). Many studies to elucidate the transport mechanism of MOP transporters were carried out using the lipid II flippase MurJ as a prototype ( 29 , 39 , 40 , 42 , 43 ). The prevailing model is that the two pseudosymmetrical bundles of transmembrane helices adopt a V-shaped configuration.…”

“…Photo-cross-linking and biotin-tagging experiments provided evidence that the substrate transport is coupled with the transition between the inward-open and outward-open conformations ( 40 ), although the electron density of the cargo could not be unambiguously assigned in the crystal structure ( 39 ). Nevertheless, how the interaction between the precursor and the flippase results in the conformational change is still unclear.…”

Lipopolysaccharide O-antigens—bacterial glycans made to measure

“…The initial structures of two MurJ homologs in inwardfacing conformations revealed a two-lobed structure with a predominantly cationic lumen that may be accessed via a lateral gate (38,39). Mutagenesis, trapping of intermediates, and structures of different conformations (38)(39)(40)(41)(42) support an alternating access model, where substrate binds at the lateral entry gate and is captured in the lumen in the inward-facing conformation. The substrate is then proposed to be flipped by a "rocker switch" mechanism in transition to the outward-facing conformation, and its release is encouraged by lumen shrinkage.…”

Chemical Biology Tools for Examining the Bacterial Cell Wall

“…The methods of isolation and detection (Figures 2A and 2B) have been used in combination in vitro to study the order of addition of WTA precursors to PG intermediates (Figure 3B; this work will be discussed in detail in Radiolabeling). Finally, these methods have been used to identify and study the mechanism of Lipid II transport (Rubino et al, 2020) as discussed in detail in the next section.…”