Biology subjects

Senes, A.

Publications and source records attributed to Senes, A..

3 recordsLinked to original sources

bioRxiv2022

A model of the interactions between the FtsQLB and the FtsWI peptidoglycan synthase complex in bacterial cell division

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.

biochemistry↗

bioRxiv2022

Thermodynamic analysis of GASright dimerization supports a model in which stability is modulated by weak hydrogen bonding and van der Waals packing

The GASright motif, best known as the fold of the glycophorin A transmembrane dimer, is one of the most common dimerization motifs in membrane proteins, characterized by its hallmark GxxxG-like sequence motifs (GxxxG, AxxxG, GxxxS, and similar). Structurally, GASright displays a right-handed crossing angle and short inter-helical distance. Contact between the helical backbones favors the formation of networks of weak hydrogen bonds between C-H carbon donors and carbonyl acceptors on opposing helices (C-H***O=C). To understand the factors that modulate the stability of GASright, we previously presented a computational and experimental structure-based analysis of 26 predicted dimers. We found that the contributions of van der Waals packing and C-H hydrogen bonding to stability, as inferred from the structural models, correlated well with relative dimerization propensities estimated experimentally with the in vivo assay TOXCAT. Here we test this model with a quantitative thermodynamic analysis. We used FRET to determine the free energy of dimerization of a representative subset of 7 of the 26 original TOXCAT dimers using FRET. To overcome the technical issue arising from limited sampling of the dimerization isotherm, we introduced a globally fitting strategy across a set of constructs comprising a wide range of stabilities. This strategy yielded precise thermodynamic data that show strikingly good agreement between the original propensities and {Delta}G{degrees} of association in detergent, suggesting that TOXCAT is a thermodynamically driven process. From the correlation between TOXCAT and thermodynamic stability, the predicted free energy for all the 26 GASright dimers was calculated. These energies correlate with the in silico {Delta}E scores of dimerization that were computed on basis of their predicted structure. These findings corroborate our original model with quantitative thermodynamic evidence, strengthening the hypothesis that van der Waals and C-H hydrogen bond interactions are the key modulators of GASright stability. Secondary AbstractWe present a thermodynamic analysis of the dimerization of the GASright motif, a common dimerization motif in membrane proteins. Previously, we found that the stability of GASright is modulated by van der Waals packing and weak hydrogen bonds between C-H carbon donors and carbonyl acceptors on opposing helices. The experimental dimerization propensities were obtained with an in vivo assay. Here we assess this model quantitatively by measuring the free energy of dimerization of a subset of the original dimers. The thermodynamic data show strikingly good agreement between the original propensities and their {Delta}G{degrees} of association, confirming the model and strengthening the hypothesis that van der Waals and C-H hydrogen bond interactions are the key modulators of GASright stability.

biophysics↗

bioRxiv2021

Nonideal polar residues in the core of the coiled coil of FtsLB are critical for modulating its stability and activation

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off or on state which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that 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 a "detuned" structural element. In addition, we identified suppressor mutations 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. 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 hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.

biochemistry↗