Abstract
The bacterial protein flagellin is the sole ligand of the innate immune receptor Toll- like receptor 5 (TLR5). Flagellins with strong agonism bind TLR5 at their D1 and D0 domains, while poor agonist “silent” flagellins bind with D1 only. However, D0-TLR5 interactions insufficiently explain the silent phenotype. Here, we characterize the D1 domain binding kinetics of the silent flagellin RhFlaB compared to the strong canonical agonist StFliC. Using Surface Plasmon Resonance, we show that the RhFlaB-D1 binds more strongly than StFliC-D1, but forms shorter-lived complexes. Cryo-EM analysis of RhFlaB showed its D1 primary interface (PI) contains two distinct hydrophobic pockets, which should facilitate rapid association, while its D1’s secondary interface (SI) is dominated by negatively charged residues, which likely impede residue-residue interactions. Comparisons with an existing StFliC-D1 structure suggest that strong agonism requires strong binding at both the PI and SI of the D1, and silent flagellins evade detection partly through weaker SI interactions. These findings offer a foundation for designing targeted interventions that can modulate TLR5 activity to influence immunity, inflammation, or even gut microbiome composition.
Competing Interest Statement
The authors have declared no competing interest.
