Open AccessDetection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling
Author(s) -
Frederick A. Rubino,
Aurelio Mollo,
Sujeet Kumar,
Emily K. Butler,
Natividad Ruiz,
Suzanne Walker,
Daniel Kahne
Publication year - 2020
Publication title -
journal of the american chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.115
H-Index - 612
eISSN - 1520-5126
pISSN - 0002-7863
DOI - 10.1021/jacs.9b12185
Subject(s) - lipid ii , chemistry , flippase , peptidoglycan , conformational change , lipid a , biophysics , biochemistry , bacterial cell structure , mutant , lipid bilayer , transporter , transport protein , substrate (aquarium) , enzyme , bacteria , membrane , phospholipid , phosphatidylserine , biology , gene , genetics , oceanography , geology
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.