Structural Basis of TLR5-Flagellin Recognition and Signaling

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Science  17 Feb 2012:
Vol. 335, Issue 6070, pp. 859-864
DOI: 10.1126/science.1215584

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  1. Fig. 1

    TLR5-FliC interactions and mutational studies. (A) Native EMSA (upper panel) and size exclusion chromatography (lower panel) reveal formation of a 1:1 TLR5-N14VLR/CBLB502 heterodimer. (B) TLR5-ECD and TLR5-N14VLR display comparable binding for CBLB502, as revealed by a competition assay (IC50 values of 67 ± 4 pM and 139 ± 28 pM, respectively, in the presence of 90 pM CBLB502; errors are SD) using HEK293 cells that express NF-κB–luciferase reporter and hsTLR5. Data are expressed as means ± SD (n = 3). (C) Mutational studies using dimerization interface-disruption mutants (CBLB502-DIM1 and -DIM2) and D0-deletion mutant (CBLB502-ΔD0) demonstrate that both the dimerization interface of the complex structure and FliC D0 domain contribute to formation of an active signaling complex. TLR5 primary binding efficiency was analyzed by a competitive FP assay where TLR5-N14VLR interaction with fluorescein-labeled CBLB502 was inhibited by unlabeled CBLB502 or its mutants. Relative primary binding efficiency was derived from IC50 ratio of CBLB502 to mutants. TLR5 signaling was assessed in an NF-κB–dependent luciferase reporter assay, and relative signaling efficiency was assessed by the EC50 ratio of CBLB502 to mutants. Smaller ratios correspond to weaker (or less efficient) binding or signaling. (D) A competitive FP assay (left) and an NF-κB–dependent luciferase reporter cell assay (right) of CBLB502 and its mutants shown in (C). FP assay results are representative of two independent experiments (left) and cell assay data are expressed as means (n = 2) with SD below 2000 RLU (right).

  2. Fig. 2

    Overall structure of the 2:2 TLR5-N14VLR/FliC-ΔD0 complex. (A) TLR5-N14 interacts with FliC-ΔD0 into a 2:2 quaternary complex structure that organizes two TLR5 molecules in a tail-to-tail orientation where their C-terminal regions are disposed in the center of the complex. The 2:2 complex consists of two copies of 1:1 complex, 1:1 TLR5-N14VLR/FliC-ΔD0 (ribbons: yellow, TLR5-N14; green, VLR; gray, FliC D1-D2), and 1:1 TLR5-N14VLR′/FliC-ΔD0′ (ribbons with translucent surface: orange, TLR5-N14′; brown, VLR′; magenta, FliC′ D1-D2). The prime denotes that the molecule or residue comes from the second 1:1 complex in the 2:2 assembly. The 1:1 complex formation is mediated by the primary binding interface (A, B, A′, and B′) and its homodimerization to the 2:2 complex is mediated by the secondary dimerization interface (α, α′, and β). For clarity, only the TLR5-N14 and FliC D1 domain are shown in the lower panel. (B) The TLR5-N14 structure of the complex. Interface residues are color-coded according to the color scheme indicated. Two disulfide bonds and four N-linked glycans are represented by black and orange sticks, respectively. Four N-linked glycans are scattered over the TLR5 surface but are not involved in TLR5-FliC interfaces. LRR7 and LRR9 are atypically long (with 32 and 36 residues, respectively) in contrast to the other LRRs (23 to 27 residues) and protrude as long loops (fig. S3). The ascending lateral surface of the LRR domain refers to the region connecting the C-terminal end of the concave surface to the N-terminal end of the convex surface in each LRR module (36). (C) The FliC D1-D2 structure observed in the complex. Each α helix is labeled according to previous nomenclature (37).

  3. Fig. 3

    The primary binding interface of the TLR5-FliC 1:1 complex. (A) Residues in primary interfaces A (bottom) and B (right) are shown in sticks (TLR5, green; FliC, light blue) on the 1:1 TLR5-FliC complex (TLR5, yellow ribbon; FliC, gray ribbon). The protruding loop of TLR5 LRR9 is highlighted in magenta. H bonds and salt bridges are represented by dashed lines. TLR5 residues are underlined to distinguish them from FliC residues. Amino acid abbreviations: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; N, Asn; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr. (B) The LRR9 loop forms a groove that provides the major FliC-binding site. FliC Arg90 is deeply inserted into the groove and makes four H bonds with carbonyl oxygens of TLR5 Tyr267, Gly270, and Ser271 (38). FliC Glu114 buttresses and orients the Arg90 side chain toward the groove via H bonds, and also forms H bonds with TLR5 Asn277. The bottom of the groove is constructed from the main chains of Gly270 and Ser271, and its surrounding wall is decorated by eight LRR9 residues (Tyr267, Asn268, Ser272, His275, Thr276, Asn277, Phe278, and Lys279) and an LRR10 residue (Lys303). The LRR9 loop groove is shown in the orange surface; interacting residues are labeled (green labels for residues that engage only main chain in the interaction with FliC; white labels for residues that engage side chains in the interaction). FliC residues that interact with the LRR9 loop groove are shown in cyan sticks. Intermolecular and intramolecular H bonds are represented by black and cyan dashed lines, respectively.

  4. Fig. 4

    Secondary dimerization interface in the 2:2 TLR5-FliC assembly. Two of the 1:1 TLR5-FliC complex homodimerize to the 2:2 complex using dimerization interfaces α, α′, and β. The overall 2:2 complex is shown in the center, and enlarged dimerization interfaces α and β are shown in the left and right panels, respectively (TLR5′ residues, brown sticks; TLR5 residues, green sticks; FliC residues, lilac sticks). In interface α, TLR5′ Asp381′, which is conserved as an acidic residue (Asp or Glu) in all TLR5 orthologs, forms three H bonds with FliC Gln128, Gln130, and Lys135. Interface β is created by van der Waals interactions among two sets of three equivalent aromatic residues (Phe273, Phe351, and His375 of TLR5 and TLR5′), which creates a largely hydrophobic core (red dotted circle) that is conserved in all other TLR5 sequences (Phe at residue 273, Leu at residue 351, and His at residue 375; see fig. S3), and by four H bonds (Arg377-Asn350′, Arg377-Tyr373′, Arg377′-Asn350, and Arg377′-Tyr373).