Structure of the heterotrimeric membrane protein complex FtsB-FtsL-FtsQ of the bacterial divisome

Abstract

The synthesis of the cell-wall peptidoglycan during bacterial cell division is mediated by a multiprotein machine, called the divisome. The essential membrane protein complex of FtsB, FtsL and FtsQ (FtsBLQ) is at the heart of the divisome assembly cascade in Escherichia coli. This complex regulates the transglycosylation and transpeptidation activities of the FtsW-FtsI complex and PBP1b via coordination with FtsN, the trigger for the onset of constriction. Yet the underlying mechanism of FtsBLQ-mediated regulation is largely unknown. Here, we report the full-length structure of the heterotrimeric FtsBLQ complex, which reveals a V-shaped architecture in a tilted orientation. Such a conformation could be strengthened by the transmembrane and the coiled-coil domains of the FtsBL heterodimer, as well as an extended β-sheet of the C-terminal interaction site involving all three proteins. This trimeric structure may also facilitate interactions with other divisome proteins in an allosteric manner. These results lead us to propose a structure-based model that delineates the mechanism of the regulation of peptidoglycan synthases by the FtsBLQ complex.

Fig. 1. The FtsB-FtsL-FtsQ (FtsBLQ) complex in the bacterial divisome and its overall structure.

a A schematic view of the recruitment of FtsBLQ to the divisome in E. coli. Divisome proteins are shown as circles: FtsB (blue); FtsL (pink); FtsQ (yellow); the upstream (black) and downstream (white) proteins of FtsBLQ. The inner and outer membranes and the periplasm are shown in grey, light blue, and orange layers. b A cartoon model of the molecular relationship between key divisome proteins at the division site: FtsBLQ (blue, pink, and yellow), FtsK (only TM domain shown, wheat), PBP1b (green), FtsW-FtsI (cyan and purple), FtsN (grey) and FtsA (orange). Solid black arrows/lines indicate the activating/inhibitory regulations. Dashed lines with arrows indicate the recruitment. The inner membrane (IM) is drawn as a grey lipid bilayer, and peptidoglycan (PG) sugars as hexagons. c Overall structure of the FtsBLQ complex as an inverted “V” shape, showing FtsB (blue), FtsL (pink), and FtsQ (yellow) in a stoichiometry of 1:1:1, with each domain accordingly labeled. All secondary structural elements are numbered sequentially. Missing stretches of residues, including residues 90–103 of FtsB; 1–39 of FtsL; 1–21, and 259–276 of FtsQ, are drawn as dashed lines. d Domain organization of FtsB, FtsL, and FtsQ with dash lines indicating the observed interactions between each other within the complex. The TM, the coiled coil, the interaction sites, the N- and C- termini, and the start and end of each structure element are also labeled. e The length difference between the periplasmic parts of FtsBL and FtsQ is shown in the same view as (c) - left or rotated for 90° - right. f The tilted FtsBLQ structure that fits all TM domains in the membrane, in the same views as in (e). In c–f the new FtsB-FtsQ interface is highlighted with an orange star.

Fig. 2. Structural and functional studies of the transmembrane and the coiled-coil domains of the FtsBLQ heterotrimer.

a Potential interacting residues between FtsB (blue), FtsL (pink) with FtsQ (grey) from its symmetry mate at the transmembrane regions. Residues are shown in sticks and labeled in according colors. b Phenotypes of the ftsB-knock-down strain by CRISPRi and its complementation by ftsB^WT, ftsB^Q16A, ftsB^W20A or ftsB^Q16A-W20A mutants. PH: Phase contrast; Blue DAPI: chromosome stain; Purple FM4-64: membrane stain. Arrows indicate the lysed cells (red), unsegregated chromosome (cyan), lack of septum (magenta), and membrane patches (orange). Scale bar, 5 μm (middle column) and 1 μm for enlarged views of the white box. c, d The cell length (c) and cell width (d) of the ftsB^WT- or mutant-complemented cells as in (b). The comparison was performed by One-way ANOVA test. Data are presented as median (shown on the bottom) with an interquartile range. FtsB^WT: n = 564; FtsB^Q16A: n = 521, P = 0.0001 (width); FtsB^W20A: n = 543, P < 0.0001 (length, width); FtsB^Q16A-W20A: n = 522, P < 0.0001 (length), P = 0.0011 (width). e The ²²GxxG²⁵ linker region at the connection between the TM and periplasmic parts of FtsB. Residues are drawn as sticks and labeled. f Interacting residue pairs of the heterodimeric FtsBL coiled coil. The Gly-rich linker (GxxG) of FtsB is also labeled. The stammer insertion (red) is shown in FtsL, and the core a and d positions of each heptad are shown as Cα spheres. Polar (cyan) and nonpolar (orange) residues are in sticks and colored accordingly. g The primary sequence of FtsB and FtsL in the coiled-coil region with a and d heptad residues and the stammer insertion colored as in (f).

Fig. 3. The C-terminal interaction site I of FtsB, FtsL, and FtsQ forms an extended β-sheet.

a Overall structure of the FtsBLQ complex highlighting the C-terminal interaction site I in a box. FtsB: blue, FtsL: pink, FtsQ: yellow. b An enlarged view of the detailed interface as in the box in (a), showing the interacting residues in sticks and hydrogen bonds in blue dashed lines. Residues that affect complex formation are labeled in red. c A rotated view (120°) of (b) showing primarily hydrophobic interactions. d The ratio of FtsQ over FtsBL in the three-protein co-purification with different mutations as listed (Supplementary Fig. 2d). The ratio of FtsQ over FtsBL in the wild-type complex is defined as 100%. Results are the mean intensity values of SDS-PAGE bands from three biological replicates. The comparison was performed by One-way ANOVA test. Data are presented as mean with SD. P = 0.0054 (FtsQ^Y248W), P = 0.0044 (FtsB^R72A). e Complementation of the ftsB-knock-down strain by ftsB^R72A mutant, showing the elongated cell shape. PH: Phase contrast; Blue DAPI: chromosome stain; Purple FM4-64: membrane stain. Arrows indicate the unsegregated chromosome (cyan), void of chromosome (white), lack of septum (magenta), and membrane punctate (orange) and the lysed cell (red). Blue: chromosome stain; Purple: membrane stain. Scale bar, 5 μm (middle column), and 1 μm for enlarged views of the white boxes. f The cell length (left) and cell width (right) of the ftsB^WT- or mutant-complemented cells as in (e). Comparison was performed by two-tailed t-test. Data are presented as median (shown on bottom) with interquartile range. Sample size n = 564 (FtsB^WT), n = 552 (FtsB^R72A). P < 0.0001 (length), P = 0.0001 (width).

Fig. 4. Key functional residues in the interaction site II and a hypothetic model for the regulation of PG synthases.

a Overall structure of the FtsBLQ complex highlighting the interaction site II in a box. b An enlarged view of the box in (a), labeling two short helices of FtsB (α2) and FtsL (α3) and the coiled coil, with interacting residues shown in sticks and hydrogen bonds in blue dashed lines. c Key residues of the Constriction Control Domain (CCD, green) and the Activation of FtsW-FtsI Domain (AWI, purple) overlap with the interaction site II. FtsB-FtsQ interface: orange star. d Distances measured from the membrane plane to the position of CCD and AWI residues of the FtsBLQ complex (middle, ribbon), to the upper side of the TG domain of PBP1b (5HLB, left, surface), and to the interacting residues in FtsW-FtsI (modeled based on 6PL5, right, surface). FtsB: blue; FtsL: pink; FtsQ: yellow; PBP1b: green; FtsW: cyan; FtsI: purple; CCD: green, Cα spheres; and AWI: purple, Cα spheres. The largest possible distance of the key residues in FtsWI was measured by flipping the periplasmic domain of FstI for 90° upwards. The regulatory relationship between them is shown below the molecules. e Crystal structures of the FtsB^E56ALQ (light blue) and FtsB^D59HLQ (blue) complexes superimposed with the FtsB^WTLQ (dark blue), showing the displacement of the α2-α3 linker in FtsB. FtsL and FtsQ: white. f The cell length (left) and width (right) of the complemented cells by ftsB^WT, ftsB^E56A, or ftsB^D59H. Median (shown on bottom) with interquartile range is shown. FtsB^WT: n = 564; (FtsB^E56A: n = 535, P < 0.0001 (length); FtsB^D59H: n = 534, P < 0.0001 (width). g–i A hypothetical model for the regulation of PG synthases by FtsBLQ. g The mutation in the AWI domain leads to the impaired interaction and activation of FtsWI (dashed line of the activation arrow). h The mutation of FtsB^E56A and FtsB^D59H in the CCD domain may lead to a potential conformational change to enhance the activation on FtsWI (solid line of the activation arrow) and impair the inhibition on PBP1b (dashed line of the inhibition arrow), with an overall shift towards the activating state. i The recruitment of FtsN triggers constriction through FtsA and FtsBLQ and activates both FtsWI and PBP1b, which is mimicked by CCD mutations.