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Detection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling
journal contribution
posted on 2020-03-11, 18:42 authored by Frederick
A. Rubino, Aurelio Mollo, Sujeet Kumar, Emily K. Butler, Natividad Ruiz, Suzanne Walker, Daniel E. KahneBacterial
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.