Biophysical Society Thematic Meeting | Ascona 2026

Mechanobiology of Infection

Poster Abstracts

5-POS Board 5 ELUCIDATING THE ROLE OF THE PERIPLASMIC LINKER DOMAIN OF TONB IN INSIDE-OUT ENERGY TRANSDUCTION IN DIDERM BACTERIA David Brockwell 1 ; Ben Rowlinson 1 ; Emanuele Paci 2 ; 1 University of Leeds, Leeds, United Kingdom 2 University of Bologna, Department of Physics and Astronomy, Bologna, Italy Many scarce nutrients are actively imported into Gram negative bacteria by coupling a proton motive force (PMF)-driven motor complex in the inner membrane (TonB:ExbBD) to outer membrane TonB dependent transporter proteins (TBDTs) that act as both high affinity substrate receptors and transport channels into the periplasm. Despite their importance as virulence factors and potential delivery of therapeutics across the impermeable outer membrane, the mechanism of mechano-transduction is still unclear. TonB comprises a structured N-terminal transmembrane helix that associates with the ExbBD motor and a globular C-terminal domain that binds to the Ton box sequence of the “plug domain” of TBDTs that becomes accessible upon substrate binding. These structured regions are linked by a ~120 residue domain which is largely disordered and enriched in proline and glutamate residues. Apart from the “D-box” motif (which binds to ExbD) the function of this linker is unclear but is not simply to span the periplasm as replacement with an unrelated intrinsically disordered region (BIB) results in severely impaired activity. To address this question we analysed the conformational dynamics of the linker by NMR, hydrogen deuterium exchange mass spectrometry and single molecule FRET. We show that the linker is extended relative to a random coil sequence and is very sensitive to solution conditions. We then show by two orthogonal functional assays that replacement of only the first third of the linker sequence with glycine-serine repeats of the same length recapitulates the BIB phenotype and that other regions exhibited wild-type phenotypes. The extended conformation likely confers a tension to the chain that allows for more efficient mechano-transduction. Finally, we show by comparative proteomics, motility assays and PMF quantification, that disruption of mechano-transduction increases the resting PMF and expression of flagellar machinery. Cross-talk between flagella assembly and TonB function, suggests a mechanism to find scarce nutrients.

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