Abstract
Antimicrobial drug resistance poses a global health challenge that necessitates the identification of new druggable targets1,2,3. The essential lipid II flippase MurJ is a promising yet underexplored antimicrobial target in bacterial cell wall biosynthesis4,5,6,7. The only known inhibitors of Gram-negative (diderm) MurJ are the single-gene lysis proteins (Sgls) from the lytic single-strand RNA phages M (SglM) and PP7 (SglPP7)8,9. SglM and SglPP7 have distinct evolutionary origins and share no sequence similarity. Here we describe a common mechanism of MurJ inhibition by these phage-encoded Sgls. We determined the structures of MurJ-bound SglM and SglPP7 and discovered a third distinct MurJ-targeting Sgl from the predicted phage Changjiang3 (SglCJ3) that we also characterized structurally. Our findings demonstrate that all three Sgls evolved convergently to trap MurJ in a periplasm-open conformation through a common MurJ interface, revealing a pathway for drug design.
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Data availability
Atomic coordinates of SglM–MurJ, SglPP7–MurJ and SglCJ3–MurJ are deposited at the Protein Data Bank (PDB) with accession codes 9NU4, 9NU5 and 9NU8, respectively. Cryo-EM maps of SglM–MurJ, SglPP7–MurJ and SglCJ3–MurJ are deposited at the Electron Microscopy Data Bank (EMDB) with accession codes EMDB-49796, EMDB-49797 and EMDB-49798, respectively. Source data are provided with this paper.
Code availability
The code used for microscopy analysis of lysis morphology is available from GitHub at https://github.com/AntillonF/bleb_statistical_analysis.git.
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Acknowledgements
We thank S. Chen, G. Pinton Tomaleri and T. Brittain for assistance with cryo-EM data collection; P. Dutka, I. Yen and J. Lee for help with cryo-EM data processing and the University of Michigan Life Science Institute Cryo-EM summer workshop for cryo-EM training. We thank T. Bernhardt (Harvard Medical School) for the strain CS7 and the plasmid pCS126. We are grateful to D. C. Rees for comments on the manuscript. Cryo-EM was performed at the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Phase contrast microscopy was performed at the Beckman Institute Biological Imaging Facility at Caltech. Funding for this work was provided by National Institutes of Health grant nos. R01GM114611 (W.M.C.), R35GM136396 (R.Y.), T32GM135748 (S.F.A.), the G. Harold and Leila Y. Mathers Foundation (W.M.C.), the Chan Zuckerberg Initiative and the Center for Phage Technology at Texas A&M University, jointly sponsored by Texas A&M AgriLife.
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Contributions
W.M.C., Y.E.L. and R.Y. conceptualized the project. Y.E.L. designed expression constructs, purified all Sgl–MurJ complexes, collected and processed cryo-EM data. Y.E.L. and W.M.C. performed model building, refinement and validation. R.Y. oversaw all work to identify the target of SglCJ3. K.C. identified the MurJ-resistant mutant for SglCJ3. S.F.A. performed physiological characterization of SglCJ3. G.F.B. and Y.E.L. constructed and characterized all lysis protein variants. Y.E.L. and W.M.C. wrote the manuscript with input from all authors.
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Extended data figures and tables
Extended Data Fig. 1 Construct design and purification of Sgl–MurJ complexes.
a, Cartoon representation of TaMurJ crystal structures in inward closed (PDB:6NC6) and outward (PDB: 6NC9) states and the EcMurJ crystal structure (PDB:6CC4) in an inward closed state with the N-lobe, C-lobe, and TMs 13-14 colored (blue, green and pink, respectively). b, Crystal structure of EcMurJ BRIL fusion (EcMurJBRIL) in an inward closed state showing the full construct. The distortion of TM1 and the deleted C-terminal residues are highlighted by dashed boxes. c, Left top, schematic of co-expression constructs for the Sgls and MurJ. Left bottom, various constructs of EcMurJ in a MurJ-depletion strain. The genomic EcMurJ is controlled by an arabinose promoter. In the absence of arabinose, only cells expressing a complementing MurJ are viable, in this case under the control of an IPTG inducible promoter. The construct used for structural studies, EcMurJBRIL, complements similar to wild-type MurJ. The non-functional R24A mutant33 is included as a negative control. Right, Lysis assay comparison of the His-tagged SglM in the absence and presence of our co-expressed EcMurJBRIL, demonstrating that EcMurJBRIL can rescue lysis. d, Representative size exclusion chromatography profiles and SDS-PAGE analyses of purified Sgl–MurJ and Sgl–MurJ–Fab–Nb complexes. Molecular weight markers are indicated in KDa. The SglCJ3 band was not observed on the SDS-PAGE gel, likely due to low sensitivity. The predicted location of SglCJ3 is labeled.
Extended Data Fig. 2 Cryo-EM data processing and analysis of the SglM–MurJ complex.
a, Summary of Cryo-EM data processing workflow to obtain the SglM–MurJ complex structure as described in methods. b, Representative micrograph (left) and 2D class averages (right). c, Angular distribution of particles of the final 3D reconstruction. d, Fourier shell correlation (FSC) curved between two half maps. e, Local resolution map of the membrane region of the final Cryo-EM map colored by resolution from 3.2 Å (purple) to 4.4 Å (green). f, Cryo-EM density of each TM helix and the BRIL fusion domain of MurJ with respective coordinates shown as sticks. Residues in MurJ making contacts with the BRIL domain are highlighted in orange.
Extended Data Fig. 3 Lysis profiles of Sgl variants.
All growth curves are shown with the mean and error bars representing the standard deviation derived from n = 3 technical replicates. For all assays, ‘uninduced’ represents the wild-type Sgl. Sgl variants were induced at time 0 and the absorbance at 600 nm was monitored over time. a, Lysis profiles of key residues in SglM, SglPP7, and SglCJ3, respectively as shown in Figs. 1c, 2a, and 3f. Representative western blots of the membrane fractions after 30 min of induction are shown below each lysis plot (See uncropped blots in Supplementary Fig. 1). b, Phase contrast images of uninduced control and cells expressing representative SglM mutants (L13A and D18A) 60 min post-induction. White arrows highlight lysed cells and cells showing septal blebbing morphology. Scale bar represents 5 µm. c, Lysis profiles of mutants that have no obvious effect on lysis onset in SglM, SglPP7, and SglCJ3, respectively. Schematics of Sgl sequences are shown for each Sgl with the transmembrane domain and secondary structure indicated above. Residues are colored based on their phenotypes.
Extended Data Fig. 4 Cryo-EM data processing and analysis of the SglPP7–MurJ complex.
a, Summary of Cryo-EM data processing workflow to obtain the SglPP7–MurJ complex structure. b, Representative micrograph (left) and 2D class averages. c, Angular distribution of particles of the final 3D reconstruction. d, Fourier shell correlation (FSC) curved between two half maps. e, Local resolution map of the transmembrane region in the final Cryo-EM map colored by resolution from 3.4 Å (purple) to 4.6 Å (green). f, Cryo-EM density fit of the TM helices 1-14 of MurJ. All models are shown in sticks. Residues in MurJ making contacts with the BRIL domain are highlighted in orange.
Extended Data Fig. 5 Physiological characterization of SglCJ3.
a, Representative microscopy images of the lysis morphology after induction of SglCJ3. b, Suppression of CJ3 lysis by heterologous lipid II flippase homologs. Over-expression of Amj from B. subtilis rescued cell lysis induced by MurJ-targeting Sgls. SglKU1 does not target MurJ and KU1-induced lysis cannot be suppressed by Amj.
Extended Data Fig. 6 Genome organization of MurJ-targeting ssRNA phages.
a, Genomes of ssRNA phages (M, PP7, and CJ3) with the three core genes mat, coat, and rep (dark blue, blue, and green, respectively). Each sgl is embedded in an alternative reading frame indicated in orange. b, Sequence alignment of the rep genes from M, PP7, and CJ3. The relative locations of each Sgl sequence to the rep proteins are shown. Sequences of the dispensable N-termini of SglPP7 and SglCJ3 are not shown.
Extended Data Fig. 7 Cryo-EM data processing and analysis of the SglCJ3–MurJ complex.
a, Summary of Cryo-EM data processing workflow to obtain the SglCJ3–MurJ complex structure. b, Representative micrograph (left) and 2D class averages. c, Angular distribution of particles of the final 3D reconstruction. d, Fourier shell correlation (FSC) curved between two half maps. e, Local resolution map of the transmembrane region in the final Cryo-EM map colored by resolution from 3.2 Å (purple) to 4.4 Å (green). f, Cryo-EM density fit of the TM helices 1-14 of MurJ. All models are shown in sticks. Residues in MurJ making contacts with the BRIL domain are highlighted in orange.
Extended Data Fig. 8 Structural analysis of Sgl-bound MurJ.
a, Comparison of the three Sgl-bound MurJ structures aligned to either the N- or the C-lobe. Sgls are not shown. b, Comparison of Sgl-bound EcMurJ to the outward TaMurJ structure. Dashed boxes highlight structural differences in TM2 and TM7. c, As in b, comparison of the inward closed EcMurJ (PDB: 6CC4), TaMurJ (PDB:6NC6), and AeMurJ (PDB:7WAW) structures. d, Structure-based multiple sequence alignment based on MurJ structures in c for TM2 and TM7 of MurJ using PROSMAL3D with ClustalX coloring for residues. Sequence differences in the G/A-E-G-A motif in TM2 are highlighted.
Extended Data Fig. 9 Sequence alignment of representative Gram-negative MurJ homologs.
Structure-based multiple sequence alignment using PROSMAL3D51 with ClustalX coloring for residues. MurJ homologs are (Uniprot IDs in brackets): Escherichia coli (P0AF16), Klebsiella pneumoniae (B5XXI8), Acinetobacter baumannii (D0CEW3), Pseudomonas aeruginosa (Q9HVM2), Shigella flexneri (Q83RT5), Neisseria gonorrhoeae (Q5F648), Haemophilus influenzae (A5UIA5), Salmonella typhimurium (P37169), Vibrio cholerae (O34238). Secondary structure based on the outward-facing EcMurJ are shown above the sequence and colored as in Extended Data Fig. 1a. Residues in MurJ that contact each Sgl are labeled with *.
Supplementary information
Supplementary Fig. 1
Uncropped Western blots with total protein staining. a–c, Uncropped western blots of the membrane fractions from E. coli cells expressing indicated Sgl variant for SglM (a), SglPP7 (b) and SglCJ3 (c). Cropped regions are indicated by white boxes as shown in Extended Data Fig. 3a. Total protein on the membrane was visualized on a replicate blot using Revert 700 Total Protein Stain (LiCor) and is shown next to the corresponding blots. Molecular weight markers are indicated. d, Bar graphs showing the relative protein level in the membrane for each Sgl variant. Band intensity was quantified using densitometry normalized to total protein signals using Empiria Studio (LiCor) across at least three independent experiments. Individual data points are shown with the mean for each Sgl variant.
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Li, Y.E., Antillon, S.F., Baron, G.F. et al. Convergent MurJ flippase inhibition by phage lysis proteins. Nature (2026). https://doi.org/10.1038/s41586-026-10163-w
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DOI: https://doi.org/10.1038/s41586-026-10163-w
