| Summary: | <p>Understanding fundamental processes within bacteria is crucial to building new approaches to tackle the problem of antimicrobial resistance. In particular, the cell envelope is a key focus as it is a target for current and future antibiotics. The Gram-negative cell envelope typically comprises of an outer membrane (OM), cell wall and inner membrane (IM). The OM acts as a barrier to the outside world and can confer intrinsic resistance to antibiotics. However, the OM is non-energised and so active processes that are carried out at the OM must harness an energy source from elsewhere. One system that carries out such a process is the Tol-Pal system that works to actively maintain the integrity of the OM. Tol-Pal utilises the proton motive force (PMF) across the inner membrane to carry out this role.</p>
<p>There are five core components of the Tol-Pal system that span the cell envelope. Pal is a lipoprotein anchored into the OM that binds the peptidoglycan (PG) cell wall. TolB is a periplasmic protein that binds Pal with high affinity. When in complex with TolB, Pal can freely diffuse within the plane of the OM. The Tol-Pal system works to modulate the binding state of Pal between PG and TolB. During cell division, Pal is recruited to the septum to ensure the OM is tethered to the PG as the cell divides. The IM components TolQ-TolR are responsible for transducing the PMF to the IM effector protein TolA. It is thought that upon interaction and protonation, TolA extends across the periplasm to bind the TolB-Pal complex at the OM. TolA then relaxes back to its ground state, dissociating TolB from Pal, allowing for the deposition of Pal onto PG.</p>
<p>The work described in this study aimed to better our understanding of the IM components of Tol-Pal by integrating a range of structural, biochemical, computational, and biophysical techniques. The first focus was to elucidate the role of the second domain of TolA in extending across the periplasm to bind TolB-Pal. A combination of Far UV circular dichroism, size exclusion chromatography multi-angle light scattering, and nuclear magnetic resonance determined that the domain is helical, monomeric and has flexible N- and C-termini. Analysis of the AlphaFold2 model of the domain reveals an extended helical hairpin structure in this domain that is the length of the periplasm. Molecular dynamic simulations demonstrated the helices of this domain can interact, and provided a basis for a new mechanism by which TolA reaches the OM.</p>
<p>The assembly of the IM stator component of Tol-Pal, TolQ-TolR, was also investigated. First, extensive optimisation was carried out to identify conditions where TolQ-TolR could be purified. Following this, the stoichiometry of the complex was investigated through biochemical methods, native mass spectrometry, and cryo-EM. In addition, the assembly of TolQ-TolR was modelled through computational means and the model evaluated in relation to experimental data. The final project of the study investigated the IM interactions of the TolQ-TolR-TolA assembly through a photoactivatable crosslinking approach. The TolAHis22BPA mutation was demonstrated to be functional, but crosslinks between TolA and TolQ at this site could not be detected. Modelling of AlphaFold2 TolA into the TolQ-TolR model showed TolA binds to a groove between two transmembrane helices of TolQ. In combination, the study furthers our understanding of how the Tol-Pal IM components assemble, and suggests a new ‘helical hinge’ mechanism for how TolA operates, allowing it to bridge the periplasm and bind to TolB-Pal at the OM.</p>
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