Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

Zheng, Sanduo; Sham, Lok-To; Rubino, Frederick A.; Brock, Kelly P.; Robins, William P.; Mekalanos, John J.; Marks, Debora S.; Bernhardt, Thomas G.; Kruse, Andrew C.

doi:10.1073/pnas.1802192115

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

Journal Article · Mon Jun 11 00:00:00 EDT 2018 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1802192115· OSTI ID:1462566

Zheng, Sanduo ^[1]; Sham, Lok-To ^[1]; Rubino, Frederick A. ^[2]; Brock, Kelly P. ^[1]; Robins, William P. ^[1]; Mekalanos, John J. ^[1]; Marks, Debora S. ^[2]; Bernhardt, Thomas G. ^[1]; ^[1]

Harvard Medical School, Boston, MA (United States)
Harvard Univ., Cambridge, MA (United States)

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Finally, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: Center of Excellence for Translational Research (CETR); NIH

OSTI ID:: 1462566

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 26 Vol. 115; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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