The mechanism of the bacterial cell wall flippase MurJ
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Rubino, Frederick A.
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CitationRubino, Frederick A. 2019. The mechanism of the bacterial cell wall flippase MurJ. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractAntibiotic-resistant infections caused by Gram-negative pathogens pose a major threat to human health. New antibiotics to treat these infections are desperately needed. The bacterial cell wall, also known as peptidoglycan, is required for survival, and its biosyn- thesis has proven to be an excellent target. Lipid II, the building block of peptidogly- can, is a lipid-linked disaccharide-pentapeptide that is synthesized on the inner leaflet of the cytoplasmic membrane and then flipped outward to the periplasmic leaflet where it is polymerized and cross-linked into the cell wall. Although most peptidogly- can biosynthetic enzymes were discovered decades ago, the essential membrane protein MurJ was only recently shown to be essential for Lipid II export, though its identification as the Lipid II flippase remains controversial. MurJ is a promising new antibiotic target; exploiting it for this purpose requires quantitative assays to monitor its inhibition and a better understanding of its transport mechanism. This work establishes a direct role for MurJ in the export of Lipid II and provides evidence that it uses a rocker-switch mechanism powered by the membrane potential. These discoveries were facilitated by our development of several key techniques as well as the application of existing ones in chemical biology, crystallography, and bioinformatics.
We first found that MurJ inhibition can be quantified by measuring the accumulation of intracellular Lipid II using a biotin-tagging strategy. This strategy also allowed us to detect photocrosslinked intermediate transport states, establishing that MurJ directly exports Lipid II. To better understand the requirements for Lipid II transport, we demon- strated that MurJ is inhibited in the absence of membrane potential. Under this condition MurJ relaxes into an inactive, outward-facing conformation for which there was no reported structure. Using sequence covariation we identified a network of residues which are predicted to interact in this conformation, suggesting structural features of the outward-open state. We subsequently crystallized an engineered variant of MurJ by the lipidic cubic phase method and determined its structure to 3.1-Å resolution. Two distinct outward-open structures are contained in the asymmetric unit, together suggesting a mechanism for Lipid II export. In sum, these techniques make up a toolkit by which to further explore the mechanism of MurJ transport.
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