title: Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor.
abstract: Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation.
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia.
authors.forename: Karolina
authors.initials: K
authors.lastname: Ivičak-Kocjan
authors.forename: Gabriela
authors.initials: G
authors.lastname: Panter
authors.forename: Mojca
authors.initials: M
authors.lastname: Benčina
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D005408:Flagellin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D051198:Toll-Like Receptor 5; D005408:Flagellin