title: Identification of a sequence in human toll-like receptor 5 required for the binding of Gram-negative flagellin.
abstract: Flagellins from Gram-negative bacteria activate inflammatory cells by a toll-like receptor 5 (TLR5)-dependent signaling pathway. We have examined the interaction between flagellin and TLR5 using an in vitro binding assay. Purified recombinant His-tagged flagellin from Salmonella enteritidis bound to TLR5 in detergent lysates from COS-1 cells transiently transfected with a human TLR5 expression plasmid. Flagellins from Salmonella typhimurium and Escherichia coli also bound to TLR5. The specificity of this interaction was demonstrated by its concentration dependence and lack of TLR5 binding to a biologically inactive form of flagellin or to a His-tagged non-flagellar protein. Flagellin bound to the extracellular domain of TLR5 expressed on the surface of COS-1 cells and to a soluble, monomeric form of the extracellular domain (amino acids 1-636). Although a TLR5 extracellular domain containing amino acids 1-407 retained flagellin binding activity, binding was not evident with a TLR5 peptide encoding residues 1-386. Conversely, a peptide containing amino acid residues 386-636 retained flagellin binding. Thus it is likely that amino acids 386-407 is a binding site for flagellin. This sequence contains a putative leucine-rich repeat. These results support the conclusion that flagellin signaling via TLR5 involves a direct interaction between flagellin and a leucine-rich region in TLR5. We also show that the NH2-terminal 358 amino acids of TLR5 play an important role in its signaling activity. Our results provide, for the first time, a molecular basis for the agonist specificity of a TLR.
authors.affiliation: Department of Microbiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. smizel@wfubmc.edu
authors.forename: Steven B
authors.initials: SB
authors.lastname: Mizel
authors.forename: A Phillip
authors.initials: AP
authors.lastname: West
authors.forename: Roy R
authors.initials: RR
authors.lastname: Hantgan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002498:Centrifugation; D004755:Enterobacteriaceae / Q000737:chemistry*; D005408:Flagellin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D014162:Transfection
chemical_list: D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D005408:Flagellin