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.

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.

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 (grey). (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.

Identification of key loops for EnvC binding in the EcoFtsEX/EnvC Complex.

(A) Overlay of EcoFtsEX and EcoFtsEX/EnvC complexes, 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 rosy 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), (F), and (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).

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.

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, the presence of physiological ATP stabilizes a fully functional FtsEX complex. Subsequently, the asymmetrical and inactive EnvC approaches the relatively flexible PLD domain of FtsEX. 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.