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J. Biol. Chem., Vol. 278, Issue 26, 23624-23629, June 27, 2003

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Identification of a Sequence in Human Toll-like Receptor 5 Required for the Binding of Gram-negative Flagellin^*

Steven B. Mizel  , A. Phillip West  and Roy R. Hantgan ¶

From the Departments of Microbiology and Immunology and ^¶Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

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.

Received for publication, April 3, 2003 , and in revised form, April 22, 2003.

^* This work was supported by National Institutes of Health Grants AI38670 and AI51319. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. E-mail: smizel{at}wfubmc.edu.

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