Structural insights into peptidoglycan hydrolysis by the FtsEX system in Escherichia coli during cell division
- Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore
- Department of Crystallography and Structural Biology, Instituto de Química-Física “Blas Cabrera”,CSIC, Spain
- Center for Bioimaging Sciences, Department of Biological Sciences, National University of Singapore, Singapore
- School of Life Sciences, University of Warwick, United Kingdom
- Infectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Figures
Figure 1 with 1 supplement
Biochemical reconstitution of EcoFtsEX and EcoFtsEX/EnvC complexes.
(A) Evaluation of ATP's impact on FtsEX and FtsEX/EnvC complexes. Purified FtsEX and FtsEX/EnvC complexes undergo precipitation in the absence of ATP but form stable complexes when ATP is present during purification. S, supernatant; P, precipitate. (B) Representative negative staining image and 2D average of FtsEX samples purified in the presence of ATP, with FtsEX highlighted in green. The FtsE subunit, transmembrane domain (TMD), and periplasmic domain (PLD) of FtsX are clearly visible. (C) Assessment of ATP and ATP analogs' influence on the stability of FtsEX complex formation. ATP and ATP analogs contribute to the stabilization of the FtsEX complex. S, supernatant; P, precipitate. (D) Assessment of ATPase activity in FtsEX and FtsEX mutants, with and without EnvC. Notably, the FtsEE163QX mutants exhibit significantly reduced ATPase activity. Data represent an average of three replicates, with error bars indicating mean ± SD. Statistical analysis performed using a two-tailed unpaired t-test; ***p<0.0005. (E). Pull-down study illustrating EnvC binding to FtsEX or its ATPase mutant in the presence or absence of ATP or ATP analogs. The initial FtsEX or FtsEE163QX added to the resin contains 2 mM ATP; the ATP was subsequently removed by the wash buffer. (F) ATPase activity comparison between FtsEX and the FtsEX/EnvC complex in peptidiscs. Data represent an average of three replicates, with error bars indicating mean ± SD.
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Figure 1—source data 1
PDF file containing the full uncropped SDS-PAGE gels for Figures 1A, C, and E, with the relevant bands, samples, and treatments indicated.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig1-data1-v1.zip
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Figure 1—source data 2
Original full raw, uncropped, and unedited gel image files for Figure 1A, C, and E.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig1-data2-v1.zip
Figure 1—figure supplement 1
Size exclusion chromatography (SEC) analysis of the impact of ATP on the FtsEX complex.
(A) SEC profiles and SDS-PAGE gels depicting EcoFtsEX and EcoFtsEX/EnvC behavior in the presence of ATP. The inclusion of 2 mM ATP results in the formation of a stable complex for both EcoFtsEX and EcoFtsEX/EnvC, as demonstrated by their tight association throughout the SEC process. (B) SEC profiles and SDS-PAGE gels showcasing FtsEX complexes from Streptococcus pneumoniae (Sp), Pseudomonas aeruginosa (Pae), and Bacillus subtilis (Bs) in the presence or absence of ATP. Without ATP, FtsEX complexes tend to dissociate, while in the presence of ATP, they form stable complexes with correct stoichiometry.
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Figure 1—figure supplement 1—source data 1
PDF file containing the full uncropped SDS–PAGE gels for Figure 1—figure supplement 1A and B, with the relevant bands, collection fractions, and nucleotide conditions indicated.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original full raw, uncropped, and unedited gel image files for Figure 1—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig1-figsupp1-data2-v1.zip
Figure 2 with 3 supplements
Cryo-EM study of EcoFtsEX complex in peptidiscs.
(A) Topological diagram of FtsEX, highlighting the α helices represented as cylinders and β sheets depicted as arrows. Color scheme: FtsE (magenta and dark green), FtsX (cornflower blue), Porter of PLDFtsX (yellow), and X-lobe of PLDFtsX (green). CH: coupling helix, EH: elbow helix. (B) Left and middle panels depict the side and front views of the density map of FtsEX, with ATPγS displayed in the front view at 90% transparency. Ribbon representation of WT FtsEX in the presence of ATPγS with EM density. Color scheme: FtsE (magenta and dark green), FtsX (cornflower blue and rosy brown), Porter of PCDFtsX (yellow), and X-lobe of PLDFtsX (green). EH: elbow helix. (C) Left panel presents a rainbow representation of the PLD domain of FtsX, transitioning from blue (N-terminal) to red (C-terminal). Right panel shows a front view overlay of two PLD domains. (D) Surface representations of the PLD domains in a top-view configuration, with the Porter of the PLD colored in yellow and the X-lobe in green. In EcoFtsEX, the Porter and X-lobe are positioned facing each other.
Figure 2—figure supplement 1
Biochemical reconstitution and single-particle cryo-EM analysis of FtsEX complex.
(A) Representative cryo-EM image with several particles boxed out by yellow circles. (B) Representative 2D averages of cryo-EM particle images. The box dimension is 270 Å. (C) Image processing flowchart. The final maps of one major conformation with its overall resolutions are indicated in red. The extra density uses a dotted line to outline. (D) Angular distribution of the cryo-EM particles included in the final 3D reconstruction. (E) The Fourier shell correlation (FSC) curve: gold standard FSC between two half data maps with indicated resolution at FSC = 0.143 (FSC corrected applied). (F) Side view (contour level 0.22 in Chimera) and front view (contour level 0.1 in Chimera) show the surface cryo-EM map filtered to the estimated overall resolution and colored according to local resolution. The extra density uses a dotted line to outline.
Figure 2—figure supplement 2
Cryo-EM density of different regions of FtsEX in the structure of FtsEX complex.
(A) Cryo-EM density of different regions of FtsE, FtsX, and ATP in the structure of FtsEX complex. (B) Structural comparison of EcoFtsEX and PaeFtsEX (PDB: 8I6R), showing high structural similarity.
Figure 2—figure supplement 3
Structural comparison of PLD domain between EcoFtsEX and PaeFtsEX.
Sequence and structure alignment of PLD domain between EcoFtsEX and PaeFtsEX. Left: Topology diagram of PLD domain in EcoFtsEX. α helices are shown as cylinders, β sheets are shown as arrows, the Porter of PLDFtsX in yellow, and the X-lobe of PLDFtsX in green; Right: Structural comparison of PLD monomers between EcoFtsEX and PaeFtsEX with the structures overlaid on each other, the PLD of EcoFtsX in blue and the PLD of PaeFtsX in brown.
Figure 3 with 5 supplements
Cryo-EM structure of the EnvC-bound EcoFtsEX complex.
(A) Domain arrangements and the overall structure of FtsEX/EnvC. Left and middle panels display side and front views of the cryo-EM density maps for FtsEX-EnvC complex in the presence of ATP. The right panel presents a ribbon representation of the structural model overlaid with the EM density. In the front-view, FtsE is depicted at 90% transparency to reveal the ATP-binding site. Color scheme of EnvC: α1 (plum), lip (purple), α2 (red), RH1, RH2, and LytM domains (gray). (B) EnvC binding to the PLD domain of FtsEX is depicted with ribbon representations. Color scheme: FtsX (cornflower blue), FtsE (magenta/dark green), and EnvC (red). (C, D) Top-down views provide a detailed depiction of the interaction between EnvC and FtsEX.
Figure 3—figure supplement 1
Biochemical reconstitution and single-particle cryo-EM analysis of FtsEX/EnvC complex.
(A) Representative cryo-EM image with particles marked by yellow circles (256 pixel box size). (B) Representative 2D averages of cryo-EM particle images with box dimension (270 Å). (C) Image processing flowchart with 256 pixel box size. The final maps of one major conformation with its overall resolutions are indicated in red. (D) Angular distribution of the cryo-EM particles included in the final 3D reconstruction. (E) The Fourier shell correlation (FSC) curve: gold standard FSC between two half data maps with indicated resolution at FSC = 0.143 (FSC corrected applied). (F) The surface cryo-EM map filtered to the estimated overall resolution and colored according to local resolution (contour level: 0.19 in Chimera).
Figure 3—figure supplement 2
Cryo-EM density of different regions of FtsEX and EnvC in the structure of FtsEX-EnvC complex.
Figure 3—figure supplement 3
Structural comparison of PLD domain between EcoFtsEX/EnvC and PaeFtsEX/envC.
Left: overlay of EcoFtsEX/EnvC and PaeFtsEX/envC (PDB: 8I6S) (Xu et al., 2023). EcoFtsEX/EnvC in blue, PaeFtsEX/envC in pink. Right: Surface representations of the PLD domains of EcoFtsEX/EnvC and PaeFtsEX/envC with top-view, ribbon representations of the EnvC. The α1 of EnvC in plum, α2 of EnvC in red, the Porter of PLD in yellow, and the X-lobe of PLD in green.
Figure 3—figure supplement 4
Structural comparison of cryo-EM structure of FtsEX/EnvC complex and X-ray structure of PLD domain with bound EnvC.
(A) Structural comparison highlighting the PLD/EnvC domains in both the FtsEX/EnvC complex and the PLD/EnvC structure (PDB: 6TPI) (Cook et al., 2020). (B) Structural comparison focusing on the CCD domain of EnvC between the FtsEX/EnvC complex and the PLD/EnvC structure. The α1 helix is shown in plum, the lip region in purple, and the α2 helix in red. Notably, both the PLD domain of FtsEX and the CCD domain of EnvC exhibit a more symmetrical conformation in the FtsEX/EnvC structure.
Figure 3—figure supplement 5
Structural comparison of EcoFtsEX/EnvC and PaeFtsEX/EnvC.
(A) Superposition of EcoFtsEX/EnvC and PaeFtsEX/EnvC (PDB: 8I6S) in front and side views, revealing a minor tilting difference in the bound EnvC. (B) Structural comparison of EnvC, the PLD and TMD regions of FtsX, and the NBD between EcoFtsEX/EnvC (blue) and PaeFtsEX/EnvC (pink). While the TMD and NBD regions are largely identical, differences are observed in the CCD of EnvC and the PLD domains. In E. coli, the two X-lobes of the PLD domain are positioned more tightly, pushing further toward the plasma membrane. This corresponds to a slightly more compact conformation between the two CCD coils in E. coli compared to P. aeruginosa.
Figure 4
Identification of key loops for EnvC binding in the EcoFtsEX/EnvC Complex.
(A) Structural overlay of full-length EcoFtsEX and EcoFtsEX/EnvC complexes obtained in this study, with a focus on the conformational changes in the PLD domain induced by EnvC binding. The overall overlay of EcoFtsEX and EcoFtsEX/EnvC is depicted in ribbon form. Color scheme: FtsE (magenta and dark green), FtsX (cornflower blue and brown), Porter of PLDFtsX (yellow), and X-lobe of PLDFtsX (green). The TMD and FtsE regions are set at 60% transparency. (B) Depiction of the conformational changes in the PLD domains upon EnvC binding. (C) Side and front views illustrating the key loops involved in EnvC binding within the EcoFtsEX/EnvC complex. Key loops of FtsX, highlighted in blue, are indicated by arrows and labeled. (D) Side-view displaying the binding details between EnvC and flexible loops located in the X-lobe (G157-L171). (E–G) Top-down views demonstrating the binding details of EnvC and flexible loops positioned between TM1 and PLD (Y105-Q109) (E), PLD and TM2 (D208-L214) (F), and TM3 and TM4 (Q307-T311) (G).
Figure 5
Biochemical reconstitution and negative-staining EM study of the AmiB-bound EcoFtsEX/EnvC complex.
(A) Pull-down study illustrating the interaction between AmiB and FtsEX-EnvC or its ATPase mutant, in the presence or absence of ATP or its analogs. The initial FtsEX or FtsEE163QX added to the resin contains 2 mM ATP; the ATP was subsequently removed by the wash buffer. (B) Evaluation of ATPase activity in FtsEX, FtsEX/EnvC, and FtsEX/EnvC/AmiB complexes. Notably, the binding of AmiB does not significantly impact the ATPase activity of FtsEX/EnvC. Data represent an average of three replicates, with error bars indicating mean ± SD. Statistical analysis performed using a two-tailed unpaired t-test; ***p<0.0005. (C) SEC profile and SDS-PAGE gel of purified FtsEX/EnvC/AmiB complex reconstituted in peptidiscs. (D) Negative-staining micrograph and 2D average image of EcoFtsEX/EnvC/AmiB. (E) Cryo-EM density map of PaeFtsEX/EnvC/AmiB.
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Figure 5—source data 1
PDF file containing the full uncropped SDS-PAGE gels for Figure 5A and C, with the relevant bands, samples, and collection fractions indicated.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig5-data1-v1.zip
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Figure 5—source data 2
Original full raw, uncropped, and unedited gel image files for Figure 5A and C.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig5-data2-v1.zip
Figure 6 with 1 supplement
A working model illustrating FtsEX complex formation, recruitment of EnvC and AmiB, and the temporal regulation of PG cleavage activation in Gram-negative bacterial systems.
Initially, in the cytosol, physiological ATP stabilizes the formation of the FtsEX complex, while in the periplasm, the asymmetrical and inactive EnvC interacts with the flexible PLD domain of FtsEX, stimulating its ATPase activity. However, the precise sequence of ATP binding and EnvC recruitment in cells remains under debate. Upon complete recognition by FtsEX, the CCD domain of recruited EnvC undergoes a conformational change to a symmetrical state, involving compression of the coiled-coil domain, facilitating the release of the LytM domain. Interaction with EnvC activates FtsEX’s ATPase activity. The liberated LytM recruits amidases through direct interaction with the catalytic domain’s blocking helices, leading to the removal of the obstructing helix from the catalytic site and activation of PG cleavage. ATP hydrolysis likely triggers the release of EnvC and AmiB, allowing FtsEX to return to its normal ABC transporter state with a low basal ATPase activity. The potential interaction between FtsEX and the constriction ring component FtsA/FtsZ precisely locates the PG-cleaving complex at the septum for temporal regulation of PG cleavage.
Figure 6—figure supplement 1
Co-purification of endogenously FtsA/FtsZ with FtsEX from E. coli.
(A) Density map of FtsEX displayed at 70% transparency, shown in side and front views. Ribbon representation of FtsEX in the presence of ATPγS docked into EM density. Dotted lines outline the additional density. Color scheme: FtsE (magenta and dark green), FtsX (cornflower blue and brown), Porter of PLDFtsX (yellow), and X-lobe of PLDFtsX (green). (B) Mass spectrometry results demonstrating the presence of considerable amounts of EcoFtsA and EcoFtsZ in the purified EcoFtsEX samples. (C) EcoFtsA copurified with SpFtsEX. Left: SDS-PAGE analysis of the purified SpFtsEX fractions. Right: mass spectrometry analysis of SDS-PAGE bands from overexpressed SpFtsE and SpFtsX, along with endogenously copurified FtsA from E. coli. Yellow lines indicate sequence coverage by exclusive unique peptides. Coverage percentages of 6%, 44%, and 95% represent EcoFtsA, SpFtsX, and SpFtsE exclusive unique peptide coverage, respectively.
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Figure 6—figure supplement 1—source data 1
PDF file containing the full uncropped SDS-PAGE gel for Figure 6—figure supplement 1C, with the relevant bands and collection fractions indicated.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original full raw, uncropped, and unedited gel image file for Figure 6—figure supplement 1C.
- https://cdn.elifesciences.org/articles/94336/elife-94336-fig6-figsupp1-data2-v1.zip
Additional files
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Supplementary file 1
Supplementary tables.
(A) Oligonucleotides used in this study. (B) Plasmids used in this study. (C) Statistics of the cryo-EM structures presented in this study.
- https://cdn.elifesciences.org/articles/94336/elife-94336-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/94336/elife-94336-mdarchecklist1-v1.docx
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Structural insights into peptidoglycan hydrolysis by the FtsEX system in Escherichia coli during cell division
eLife 13:RP94336.
https://doi.org/10.7554/eLife.94336.3
