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A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin.

Andersen-Nissen E, Smith KD, Bonneau R, Strong RK, Aderem A - J. Exp. Med. (2007)

Bottom Line: Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition.These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand.These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

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Model of the TLR5 extracellular domain. (A) Ribbon representations (far left and far right) and molecular surface representations (middle left and middle right) of the best model of the TLR5 ectodomain (left) and the structure of flagellin (PDB lucu; right). The ribbon is colored sequentially from the N terminus (blue) to the C terminus (red). Amino acids important for flagellin recognition are shown on the TLR5 model in red. Amino acids on flagellin previously determined to be important for TLR5 recognition are shown in red on the flagellin structure (reference 5). (B) Ribbon representations (left) and molecular surface representations (middle and right) of the best model of the TLR5 ectodomain, oriented ∼90° to the view in A. Views 180° apart are shown in the top and bottom rows. The ribbon is colored sequentially from the N terminus (blue) to the C terminus. Molecular surface representations are colored by conservation (center: residues ≥90% similar among TLR5 sequences are green) or by electrostatic potential (right: positive, blue; negative, red). The conserved concavity and lateral patch regions are indicated with a dotted white oval and bracket, respectively. Positions of residues important for flagellin recognition are indicated with arrows. (C) Space-filling representation of the TLR5 model. Residues of the conserved concavity (white) and residues that differ between human and mouse TLR5 in the central 228–amino acid region, as well as 407 and 408 (red), are highlighted. Amino acids surrounding the concavity that were mutated are indicated with arrows.
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fig4: Model of the TLR5 extracellular domain. (A) Ribbon representations (far left and far right) and molecular surface representations (middle left and middle right) of the best model of the TLR5 ectodomain (left) and the structure of flagellin (PDB lucu; right). The ribbon is colored sequentially from the N terminus (blue) to the C terminus (red). Amino acids important for flagellin recognition are shown on the TLR5 model in red. Amino acids on flagellin previously determined to be important for TLR5 recognition are shown in red on the flagellin structure (reference 5). (B) Ribbon representations (left) and molecular surface representations (middle and right) of the best model of the TLR5 ectodomain, oriented ∼90° to the view in A. Views 180° apart are shown in the top and bottom rows. The ribbon is colored sequentially from the N terminus (blue) to the C terminus. Molecular surface representations are colored by conservation (center: residues ≥90% similar among TLR5 sequences are green) or by electrostatic potential (right: positive, blue; negative, red). The conserved concavity and lateral patch regions are indicated with a dotted white oval and bracket, respectively. Positions of residues important for flagellin recognition are indicated with arrows. (C) Space-filling representation of the TLR5 model. Residues of the conserved concavity (white) and residues that differ between human and mouse TLR5 in the central 228–amino acid region, as well as 407 and 408 (red), are highlighted. Amino acids surrounding the concavity that were mutated are indicated with arrows.

Mentions: Because the crystal structure of TLR5 has not yet been solved, we modeled the structure of the mouse and human TLR5 ectodomains based on known structures of other LRR proteins. Using consensus fold recognition, the best match was determined to be L. monocytogenes internalin A (PDB 1o6s). Modeling of LRR-containing proteins is inherently difficult (13), with the resulting overall global structure varying considerably with even small changes in individual LRR or with relative orientations of adjacent LRR. We evaluated several models (generated by varying method parameters), which resulted in solenoids of different global structure but largely uniform local structure. The model of the mouse TLR5 ectodomain (amino acids 52–615) that we present (Fig. 4 A, left) conforms well to ideal peptide and protein geometry while also generating an overall fold suited to binding large macromolecular ligands such as flagellin (Fig. 4 A, right). The model was further validated by its similarity to the recently deposited structure of the extracellular domain of TLR3 (PDB 1ziw). The real value of the TLR5 model was not in its high resolution atomic details but in guiding selection of residues for further study based on proximity to conserved surface residues in the TLR5 model. We identified amino acids that were similar among at least 90% of vertebrate TLR5 sequences and highlighted their positions on our structural model (Fig. 4 B, green). This analysis revealed two regions that were conserved among all species: an apparent concavity located on the inner surface of the solenoid structure predicted for the TLR5 ectodomain and a patch located on the lateral face of the molecule (Fig. 4 B). Both the concavity and the lateral patch were contained within the central 228–amino acid region that confers species-specific flagellin recognition.


A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin.

Andersen-Nissen E, Smith KD, Bonneau R, Strong RK, Aderem A - J. Exp. Med. (2007)

Model of the TLR5 extracellular domain. (A) Ribbon representations (far left and far right) and molecular surface representations (middle left and middle right) of the best model of the TLR5 ectodomain (left) and the structure of flagellin (PDB lucu; right). The ribbon is colored sequentially from the N terminus (blue) to the C terminus (red). Amino acids important for flagellin recognition are shown on the TLR5 model in red. Amino acids on flagellin previously determined to be important for TLR5 recognition are shown in red on the flagellin structure (reference 5). (B) Ribbon representations (left) and molecular surface representations (middle and right) of the best model of the TLR5 ectodomain, oriented ∼90° to the view in A. Views 180° apart are shown in the top and bottom rows. The ribbon is colored sequentially from the N terminus (blue) to the C terminus. Molecular surface representations are colored by conservation (center: residues ≥90% similar among TLR5 sequences are green) or by electrostatic potential (right: positive, blue; negative, red). The conserved concavity and lateral patch regions are indicated with a dotted white oval and bracket, respectively. Positions of residues important for flagellin recognition are indicated with arrows. (C) Space-filling representation of the TLR5 model. Residues of the conserved concavity (white) and residues that differ between human and mouse TLR5 in the central 228–amino acid region, as well as 407 and 408 (red), are highlighted. Amino acids surrounding the concavity that were mutated are indicated with arrows.
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fig4: Model of the TLR5 extracellular domain. (A) Ribbon representations (far left and far right) and molecular surface representations (middle left and middle right) of the best model of the TLR5 ectodomain (left) and the structure of flagellin (PDB lucu; right). The ribbon is colored sequentially from the N terminus (blue) to the C terminus (red). Amino acids important for flagellin recognition are shown on the TLR5 model in red. Amino acids on flagellin previously determined to be important for TLR5 recognition are shown in red on the flagellin structure (reference 5). (B) Ribbon representations (left) and molecular surface representations (middle and right) of the best model of the TLR5 ectodomain, oriented ∼90° to the view in A. Views 180° apart are shown in the top and bottom rows. The ribbon is colored sequentially from the N terminus (blue) to the C terminus. Molecular surface representations are colored by conservation (center: residues ≥90% similar among TLR5 sequences are green) or by electrostatic potential (right: positive, blue; negative, red). The conserved concavity and lateral patch regions are indicated with a dotted white oval and bracket, respectively. Positions of residues important for flagellin recognition are indicated with arrows. (C) Space-filling representation of the TLR5 model. Residues of the conserved concavity (white) and residues that differ between human and mouse TLR5 in the central 228–amino acid region, as well as 407 and 408 (red), are highlighted. Amino acids surrounding the concavity that were mutated are indicated with arrows.
Mentions: Because the crystal structure of TLR5 has not yet been solved, we modeled the structure of the mouse and human TLR5 ectodomains based on known structures of other LRR proteins. Using consensus fold recognition, the best match was determined to be L. monocytogenes internalin A (PDB 1o6s). Modeling of LRR-containing proteins is inherently difficult (13), with the resulting overall global structure varying considerably with even small changes in individual LRR or with relative orientations of adjacent LRR. We evaluated several models (generated by varying method parameters), which resulted in solenoids of different global structure but largely uniform local structure. The model of the mouse TLR5 ectodomain (amino acids 52–615) that we present (Fig. 4 A, left) conforms well to ideal peptide and protein geometry while also generating an overall fold suited to binding large macromolecular ligands such as flagellin (Fig. 4 A, right). The model was further validated by its similarity to the recently deposited structure of the extracellular domain of TLR3 (PDB 1ziw). The real value of the TLR5 model was not in its high resolution atomic details but in guiding selection of residues for further study based on proximity to conserved surface residues in the TLR5 model. We identified amino acids that were similar among at least 90% of vertebrate TLR5 sequences and highlighted their positions on our structural model (Fig. 4 B, green). This analysis revealed two regions that were conserved among all species: an apparent concavity located on the inner surface of the solenoid structure predicted for the TLR5 ectodomain and a patch located on the lateral face of the molecule (Fig. 4 B). Both the concavity and the lateral patch were contained within the central 228–amino acid region that confers species-specific flagellin recognition.

Bottom Line: Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition.These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand.These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the beta sheets results in TLR activation and provide a new framework for understanding TLR-agonist interactions.

Show MeSH