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. 2012 Feb 17;335(6070):859-64.
doi: 10.1126/science.1215584.

Structural Basis of TLR5-flagellin Recognition and Signaling

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Free PMC article

Structural Basis of TLR5-flagellin Recognition and Signaling

Sung-il Yoon et al. Science. .
Free PMC article

Abstract

Toll-like receptor 5 (TLR5) binding to bacterial flagellin activates signaling through the transcription factor NF-κB and triggers an innate immune response to the invading pathogen. To elucidate the structural basis and mechanistic implications of TLR5-flagellin recognition, we determined the crystal structure of zebrafish TLR5 (as a variable lymphocyte receptor hybrid protein) in complex with the D1/D2/D3 fragment of Salmonella flagellin, FliC, at 2.47 angstrom resolution. TLR5 interacts primarily with the three helices of the FliC D1 domain using its lateral side. Two TLR5-FliC 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex that is stabilized by quaternary contacts of the FliC D1 domain with the convex surface of the opposing TLR5. The proposed signaling mechanism is supported by structure-guided mutagenesis and deletion analyses on CBLB502, a therapeutic protein derived from FliC.

Figures

Fig. 1
Fig. 1
TLR5-FliC interactions and mutational studies. (A) Native EMSA (upper part) and size-exclusion chromatography (lower part) reveal formation of a 1:1 TLR5-N14VLR/CBLB502 heterodimer. (B) TLR5-ECD and TLR5-N14VLR display a comparable binding for CBLB502 as revealed by a competition assay (IC50 values of 67±4 (standard deviation; SD) pM and 139±28 (SD) pM, respectively, in the presence of 90 pM CBLB502) using NF-κB luciferase HEK293 reporter cells that express hsTLR5. Data are expressed as mean ± 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 presented using EC50 ratio of CBLB502 to mutants. The smaller ratio corresponds 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 Fig. 1C. FP assay results are representative of two independent experiments (left) and cell assay data are expressed as mean (n = 2) with SD below 2000 RLU (right).
Fig. 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, and gray FliC D1-2) and 1:1 TLR5-N14VLR′/FliC-ΔD0′ (ribbons with translucent surface; orange TLR5-N14′, brown VLR′, magenta FliC′ D1-2). 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 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 in the figure. 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 not involved in TLR5-FliC interfaces. LRR7 and LRR9 are atypically long with 32 and 36 residues, compared to other LRRs that contain 23-27 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 (39). (C) The FliC D1-2 structure observed in the complex. Each α-helix is labeled according to previous nomenclature (40).
Fig. 3
Fig. 3
The primary binding interface of the TLR5-FliC 1:1 complex. (A) Residues in primary interfaces-A (left) 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. To discriminate from FliC residues, TLR5 residues are underlined throughout the rest of figures. (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 (41). Glu114 buttresses and orients the Arg90 side chain toward the groove via H-bonds, and also 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 8 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 and interacting residues are labeled (green labels for residues that engage only main chains 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. Inter-molecular and intra-molecular H-bonds are represented by black and cyan dashed lines, respectively.
Fig. 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 middle, 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).

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