In , FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here, we present a structural analysis of the FtsLB complex, performed with biophysical, computational, and methods, that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain probably terminates near the critical constriction control domain, which might correspond to a structural transition. The presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions and that the integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

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In Escherichia coli, an important step in the divisome assembly pathway is the recruitment of the essential cell wall synthase complex FtsWI to the division site through interactions with the regulatory FtsQLB complex. Here, we investigate a key aspect of this recruitment by characterizing the structural organization of the FtsL-FtsW interaction. Mutations in the cytoplasmic and transmembrane regions of the two proteins result in cell division defects and loss of FtsW localization to division sites. We use these in vivo results to help validate the predicted interfaces from an AlphaFold2 model for the entire FtsQLBWI complex. Given the consistency between the predicted FtsQLBWI model and our current understanding of the structure and function of the complex, we further remodeled it, seeking insight into the potential structural transitions that may lead to activation of the FtsWI complex and PG synthesis. The model suggests that FtsLB serves as a support for FtsI, placing its periplasmic domain in an extended and possibly active conformation but it is also compatible with a proposed compact and possibly inactive conformation. Additionally, we reconfigure the model into an Fts[QLBWI]2 diprotomeric state, which suggests that FtsLB may act as a central hub during assembly of the PG synthesis machinery. Finally, we propose a possible role for FtsQ in activation of this machinery, potentially by acting as a gatekeeper for the interaction between the FtsL AWI region and FtsI. We propose that this gatekeeping function depends on a hinge next to the FtsLB CCD region, which has implications for the mechanisms behind the FtsLB off/on transition that is central to cell division regulation.

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The FtsLB complex is a critical regulator of bacterial cell division, acting as a switch that modulates cell wall reconstruction. Evidence indicates that FtsLB exists in either an off or on state which supports the corresponding activation state of the peptidoglycan synthase complex FtsWI. In Escherichia coli, residues within FtsLB that are critical for this activation are located in a region near the C-terminal end of the periplasmic coiled coil, raising questions about the precise role of this conserved domain in the mechanism. Here, we investigate an unusual cluster of polar amino acids occurring within the core of the coiled coil. These amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is an intentionally "detuned" structural element. In addition, suppressor mutations were identified within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. Based on energetic and sequence propensity considerations, we propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the polar amino acids participate in packing within the core, but their hydrophilic terminal moieties remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). The Y-model remains well structured during molecular dynamics simulations, unlike the I-model, and satisfies all known experimental constraints. For this reason, we propose the Y-model as the configuration of the coiled coil of FtsLB and that a shift in this architecture, dependent on its marginal stability, is involved in activating the complex during the process that triggers septal cell wall reconstruction.