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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

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.

Toll-like receptors (TLRs) play a key role in innate immunity by eliciting the first line of defense against invading pathogens (1). TLRs are membrane-bound antigen recognition receptors (2) that directly recognize highly conserved molecular structures in pathogens using their horseshoe-shaped, leucine-rich repeat (LRR) ectodomains. Each TLR recognizes a different set or subset of antigens with a distinct ligand-binding mechanism (fig. S1). Nonetheless, all known agonist-activated TLR structures form a similar dimer organization, which brings their C-terminal regions into juxtaposition so that their intracellular TIR domains can initiate the signaling cascades (36). To date, structural studies on TLR-ligand recognition have been limited to nonprotein antigens, and the structural basis for protein antigen recognition by a TLR has not been addressed.

TLR5 is the only protein-binding TLR that is conserved in vertebrates from fish to mammals (79). TLR5 responds to a monomeric form of flagellin from β- and γ-proteobacteria that constitutes the whiplike flagellar filament responsible for locomotion (8, 10, 11). Flagellin (hereafter termed FliC, corresponding to the main flagellin gene product in Salmonella) binding to TLR5 induces MyD88-dependent signaling and activates the proinflammatory transcription factor NF-κB in epithelial cells, monocytes, and dendritic cells, which results in activation of innate immune responses against flagellated bacteria (8, 1216).

The key roadblock against TLR5-FliC interaction studies has been the formidable technical challenge in expression of the mammalian TLR5 ectodomain (ECD) in a functionally active, soluble form. We overcame this challenge by testing the expression of a number of TLR5 orthologs (16, 17). Only the zebrafish Danio rerio ortholog (drTLR5-ECD) could be successfully expressed in a secreted soluble form in the baculovirus system, but with a low purification yield (<50 μg from 1 liter of culture) (18). To improve the yield, we engineered a series of drTLR5-ECD C-terminal deletion variants as chimeric proteins with the C-terminal fragment of hagfish variable lymphocyte receptor (VLR) B.61 using a strategy successfully used for the crystallization of other TLRs (4, 19). Three chimeric proteins containing 6, 12, and 14 N-terminal LRR modules (TLR5-N6VLR, TLR5-N12VLR, and TLR5-N14VLR, respectively) (fig. S2A) were expressed, purified to homogeneity, and tested for their interaction with CBLB502 (20) (fig. S2B), a pharmacologically optimized derivative of FliC from Salmonella enterica subspecies enterica serovar Dublin (sdFliC), by size exclusion chromatography and native electromobility shift assay (EMSA) (Fig. 1A). Stable 1:1 complexes with CBLB502 were obtained for TLR5-N12VLR and TLR5-N14VLR (but not TLR5-N6VLR) and were used for structural studies.

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).

The largest chimeric protein TLR5-N14VLR inhibited CBLB502-induced signaling as efficiently as full-length drTLR5-ECD in a competitive assay using human embryonic kidney (HEK) 293 cells stably expressing NF-κB–luciferase reporter and human TLR5 (hsTLR5) (Fig. 1B). The observed median inhibitory concentrations (IC50 values) for both derivatives of drTLR5 (67 and 139 pM; Fig. 1B) were in the same range as the EC50 of CBLB502-hsTLR5 signaling efficiency (77 pM) measured in the same reporter system (Fig. 1, C and D). Although none of these values can be interpreted as a true dissociation constant, this observation supports the relevance of drTLR5 as a model for hsTLR5 interactions with FliC.

To provide the structural basis for TLR5-FliC interaction, we determined the crystal structures of TLR5-N12VLR and a complex of TLR5-N14VLR with a D0 deletion variant of sdFliC (sdFliC-ΔD0; fig. S2B) at 2.83 and 2.47 Å resolutions, respectively (21) (Fig. 2A and table S1). In the TLR5-FliC complex, the FliC D1 domain is located on the lateral side of TLR5, forming an extensive primary binding interface that defines a 1:1 heterodimer. Two heterodimers further oligomerize to a symmetric 2:2 complex where TLR5 from the first heterodimer makes additional, weaker interactions with both FliC′ and TLR5′ from the second heterodimer, forming a secondary dimerization interface. This TLR5-FliC interaction mode is completely different from other TLR interactions with nonprotein ligands with respect to stoichiometry, ligand arrangement, and binding interfaces (16). Nonetheless, flagellin binding results in assembly of two TLR5s in a tail-to-tail organization, which juxtaposes the C-terminal tail regions of TLR5-ECD for signaling by analogy with other agonist-bound TLRs (fig. S1). Thus, the TLR5-N14VLR/FliC-ΔD0 structure illustrates distinct, as well as conserved, features relative to other TLRs for receptor activation.

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).

TLR5 adopts a single-domain LRR structure that consists of an N-terminal β-hairpin capping motif (LRRNT), 13 complete LRR modules (LRR1 to LRR13), and two residues from LRR14 (22) (Fig. 2B and fig. S3). The concave surface displays a smooth, curved β-sheet structure that is formed from two antiparallel β strands of LRRNT and 13 parallel β strands of LRR modules. However, the convex surface is less regular, with an assortment of helices and extended structures. Comparison of TLR5 with other known TLR structures indicates that TLR5 can be better categorized as a TLR3-like LRR structure than as a TLR4- or TLR2-like structure, consistent with the evolutionary closeness of TLR3 and TLR5 (23) (fig. S4).

Two of the three FliC-ΔD0 domains, D1 and D2, could be modeled in the complex structure (24) (fig. S5), but only D1 is involved in direct interaction with TLR5 (Fig. 2C). The D1 domain consists of four elongated segments that vertically assemble into a long, largely helical rod. The four segments correspond to two N-terminal α helices (αND1a and αND1b), one C-terminal α helix (αCD1), and a segment of β hairpin and extended structure that connects αND1b to the D2 domain (Fig. 2C and fig. S6).

The extensive primary binding interface responsible for the specific recognition of FliC by TLR5 is formed between the ascending lateral surface of TLR5 LRRNT to LRR10 and one side of the three long α helices of FliC D1 with ~1320 Å2 of buried accessible surface area (b.s.a.) on each side (Fig. 3A). This interface can be described as two adjacent, but spatially separated surfaces, interfaces A and B, and includes residues previously proposed as TLR5 binding sites by a mutational study (11) (fig. S7). Moreover, the FliC interface coincides with residues involved in FliC oligomerization in the flagellar filament structure and is therefore well conserved across species of β- and γ-proteobacteria (figs. S6 and S8). These observations support the hypothesis that TLR5 evolved to target a common molecular pattern of FliC that is functionally crucial for flagellar assembly and hence is evolutionarily constrained (11).

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.

Interface A is formed between the concave and ascending sides of TLR5 LRRNT to LRR6 and the C-terminal half of FliC αCD1 with ~530 Å2 b.s.a. (Fig. 3A, bottom, and fig. S9). Interface A narrows from LRRNT toward LRR6, with LRRNT to LRR2 providing three or four residues from both concave and ascending sides, and LRR3 to LRR6 contributing one or two residues only from the ascending side. Interaction in interface A is mainly hydrophilic, with five H bonds and three salt bridges (table S2).

Interface B is located on the ascending lateral surface of the central LRR modules (LRR7 to LRR10) of TLR5 and on the upper part of FliC αND1a and αND1b (Fig. 3A, right) and constitutes ~60% (~790 Å2) of the primary interface (fig. S9, A and B). Interface B is also mainly hydrophilic, with 12 H bonds, and is primarily located at a protruding loop of LRR9 that undergoes structural changes upon FliC binding (fig. S10, A and B). In the TLR5-N12VLR structure, the LRR9 loop is highly flexible, as indicated by high B values and poor electron density, but upon FliC binding, the loop undergoes structural rearrangement to form a well-defined groove (Fig. 3B and fig. S10A) that accommodates the absolutely conserved FliC residues Arg90 and Glu114 (fig. S6). The groove side and rim reinforce FliC binding through diverse H bonds and van der Waals interactions with eight FliC residues at the C-terminal end of αND1a and N-terminal half of αND1b. This groove is responsible for half of the primary-interface H bonds and ~35% of b.s.a. The functional importance of the LRR9 loop was further demonstrated by an LRR9-loop deletion that severely impaired FliC binding in size exclusion chromatography, a competitive fluorescence polarization (FP) assay (by at least three orders of magnitude), and competitive cell-based signaling assays (by two to three orders of magnitude) (fig. S10, C to F). This loop is highly conserved in all species and likely forms the hot spot in the TLR5-FliC interaction (25).

The secondary dimerization interface consists of three surfaces: interface α (TLR5′-FliC), its two-fold symmetry–related interface α′ (TLR5-FliC′), and interface β (TLR5-TLR5′); no contacts are made between FliC molecules (26) (Fig. 4). Dimerization interface α is located on the convex side of TLR5′ LRR12/13 and on the FliC C-terminal αND1b region and following loop at the bottom of the D1 domain (Fig. 4, left). Dimerization interface β is formed by TLR5 residues mainly on the ascending lateral side of LRR12/13 (Fig. 4, right). The dimerization interface is relatively limited with a total of ~550 Å2 b.s.a. on each 1:1 complex (α, ~130 Å2; α′, ~130 Å2; β, ~290 Å2), but may be greater in the full-length TLR5-FliC interaction (16). This dimerization interface exhibits high sequence conservation among both vertebrate TLR5 orthologs and FliC orthologs from diverse bacteria (figs. S3, S6, and S11), and high shape complementarity (shape correlation statistic Sc, 0.64 to 0.76) (27), underscoring its functional importance in the secondary dimerization of TLR5-FliC (28).

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).

We used structure-guided mutagenesis to assess whether the observed dimerization interface contributes to TLR5 activation for downstream NF-κB signaling. A series of CBLB502 derivatives with the proposed dimerization surface disrupted by point mutations and/or deletions were tested in two assays: (i) a competitive FP assay to assess primary binding for drTLR5, and (ii) an NF-κB–luciferase reporter assay to assess signaling activity that results from both primary binding and secondary dimerization. The latter assay with hsTLR5 in HEK293 cells also provided an opportunity to further validate the drTLR5 structure as a surrogate for hsTLR5. The binding and signaling data from the CBLB502 mutants (Fig. 1, C and D, and table S3) support the proposed contribution of the dimerization interface to signaling. Dimerization interface mutant 1 (DIM1; Arg124 → Asp, Gln128 → Ala, Gln130 → Ala, and Lys135 → Ala mutations in αND1b at the dimerization interface α, Fig. 4) shows a factor of ~30 decrease in signaling, but essentially no decrease in binding (factor of ~3). For the second mutant (DIM2; deletion of residues 126 to 128 and Thr129 → Gly, Gln130 → Gly, and Lys135 → Glu mutations to disrupt αND1b and connecting loop structures and introduce charge repulsion), the disruption in signaling efficiency is even more pronounced (a factor of ~800 decrease, versus only a factor of 9 decrease in binding). In the control mutant CBLB502-PIM (with Gln89, Arg90, and Gln97 mutated to Ala in the primary interface), comparable decreases in primary binding (by a factor of 450) and signaling efficiency (by a factor of ~120) were observed (29). Decoupling of antigen binding and receptor signaling was also observed for CBLB502-ΔD0, used as a control, where a minor (factor of 2 to 3) change in binding affinity both for TLR5-N14VLR and TLR5-ECD contrasts with a factor of ~1000 decrease in signaling efficiency (Fig. 1, C and D, and fig. S12). Thus, both dimerization interface mutants (CBLB502-DIM1 and -DIM2) clearly correlate in activity with the signaling-impaired CBLB502-ΔD0 mutant rather than with the primary binding–impaired CBLB502-PIM mutant.

Our results indicate that the D1 domain makes substantial contributions to both high-affinity binding and TLR5 signaling (16), whereas D0 contributes to TLR5 activation, but has no or little effect on binding. Furthermore, the TLR5-FliC structure and unique features of ligand recognition and activation provide a template to enhance the therapeutic activity of the radioprotective drug (CBLB502) or flu vaccine (VAX102) in clinical trials, as well as to design new vaccine adjuvants or antagonistic therapeutics for hyperinflammatory diseases.

Supporting Online Material

www.sciencemag.org/cgi/content/full/335/6070/859/DC1

Materials and Methods

SOM Text

Figs. S1 to S14

Tables S1 to S3

References (3954)

References and Notes

  1. There are 10 functional human TLRs (TLR1 to 10) and 12 functional mouse TLRs (TLR1 to 9, TLR11 to 13).
  2. This tail-to-tail TLR dimerization is believed to be a prerequisite for signal transduction, consistent with lack of signaling by antagonist-bound TLR 1:1 complexes and a head-to-head TLR dimer of a TLR4 regulator, RP105 (19, 3032).
  3. See supplementary material on Science Online.
  4. Human, mouse, frog, trout, and zebrafish TLR5 ectodomains were screened for expression in a baculovirus system. In addition, human and mouse TLR5 ectodomains were also tested in an HEK293 cell expression system.
  5. Zebrafish contains two highly homologous tlr5 genes, tlr5a and tlr5b. Previous tlr5a/b knockdown embryo experiments showed that TLR5a/b are responsible for FliC-specific immune responses (7). TLR5b was used for this study.
  6. CBLB502 contains only D0 and D1 domains from sdFliC, which consists of four domains (D0, D1, D2, and D3). CBLB502 activates TLR5 as FliC and protects the hematopoietic system and gastrointestinal tissues from radiation-induced damage, creating opportunities for therapeutic applications (33).
  7. Despite the tight complex formation, cocrystallization trials of drTLR5 chimeric proteins with sdFliC and CBLB502 failed to yield diffractable crystals, most likely because of the excessive flexibility of the D0 domain, which appeared dispensable for formation of a 1:1 complex with TLR5 as assessed by binding of CBLB502-ΔD0 in a competitive FP assay (fig. S12). Although CBLB502-ΔD0 also failed to yield diffractable cocrystals, sdFliC-ΔD0 in complex with TLR5-N14VLR was successful in producing diffraction-quality crystals.
  8. The TLR5 structures in TLR5-N12VLR and FliC-bound TLR5-N14VLR superimpose well, except for the protruding loop of LRR9, which undergoes substantial conformational changes upon FliC binding (Cα-RMSD without the loop, 0.56 Å). Thus, for TLR5 structure analysis, the TLR5-N14VLR structure is described unless otherwise specified.
  9. The FliC D3 domain could not be built because of poor electron density, although it exhibits an apparent molecular envelope (fig. S5).
  10. Although the protein-to-protein binding interface is generally extensive, most of the binding stability and free energy are provided by a small cluster of hotspot residues. We propose that the binding hotspot of the primary drTLR5-FliC binding interface is located in the LRR9 loop, given that deletion of the LRR9 loop almost completely abrogates FliC binding. Moreover, the LRR9 loop sequence is more conserved than other primary interface residues (fig. S3). Accordingly, the LRR9 loop is anticipated to also function as a key determinant in human TLR5 interaction with FliC. About 62% of the total interactions observed at the drTLR5 LRR9 loop would be conserved in human TLR5, with 8 out of 10 H bonds recapitulated (fig. S13). In contrast, other interface residues from LRRNT to LRR8 show lower sequence conservation, consistent with our observation that a TLR5-VLR hybrid (TLR5-N6VLR) that contains a majority of interface-A residues from LRRNT to LRR6 does not form a tight complex with FliC or CBLB502 in solution. Therefore, the network of hsTLR5-FliC interactions in this region could be somewhat different. Only ~34% of the total interactions observed at the drTLR5 region from LRRNT to LRR8 would be formed in human TLR5, with 2 out of 10 H bonds functionally conserved. The conservation of the key interactions in the TLR5-flagellin interface is supported by our mutagenesis data (PIM-mutant of CBLB502) using a reporter system with hsTLR5 (Fig. 1, C and D), as well as by highly efficient, competitive inhibition of CBLB502-induced hsTLR5 signaling by drTLR5-ECD (Fig. 1B).
  11. The secondary dimerization interaction is very weak in solution for these ectodomain constructs. The 2:2 complex was not detectable by size exclusion chromatography and native EMSA even with full-length TLR5-ECD and FliC proteins. Only a trace amount of the 2:2 complex was observed between the full-length TLR5-ECD and CBLB502 with a Kd of ~0.5 mM in analytical ultracentrifugation, which suggests the instability of the secondary dimerization in solution. Nonetheless, we anticipate that FliC-mediated TLR5 dimerization would be enhanced on the cell surface, possibly as a result of the constraint of two-dimensional movement in the membrane (34), engagement of intracellular and transmembrane domains of TLR5, or contribution of other unknown co-receptor/adaptor molecules.
  12. The secondary dimerization interface of TLR5-N14/FliC extends to interactions between VLR and VLR′ (b.s.a., ~370 Å2), between VLR and FliC′ (b.s.a., ~100 Å2), and between VLR′ and FliC (b.s.a., ~40 Å2) in the TLR5-N14VLR/FliC-ΔD0 structure. However, the VLR interactions exhibit very low shape complementarity (0.42, 0.16, and 0.04 of Sc values, respectively) and do not include any specific H bonds and salt bridges. These observations suggest that they make minimal contributions to TLR5-N14VLR/FliC-ΔD0 dimerization.
  13. Individual alanine mutations at FliC Gln89, Arg90, and Gln97 reduced TLR5 signaling by factors of 4.1, 7.4, and 5.6, respectively, in a previous study (11).
  14. Amino acid variations at groove-forming residue 271 (Ala267 in human, Pro268 in mouse, and Ser271 in zebrafish) were implicated in human versus mouse differences in the cellular response to FliC (35). The same study also showed that mutations of two TLR5 aspartate residues (Asp294 of LRR10 and Asp366 of LRR13 in human TLR5) modulated the FliC response. However, these two acidic residues are not in the TLR5-FliC interface, but are instead located in the middle of the concave β strands and are involved in intramolecular interactions with nearby serine residues and the Asn346-linked glycan, suggesting a critical role in TLR5 protein folding or stability. Thus, it is expected that the mutations would indirectly affect FliC response through modulation of TLR5 protein integrity, rather than directly through FliC binding.
  15. Acknowledgments: We thank Y. S. Choo for critical comments on the manuscript, H. Tien and D. Marciano for automated crystal screening, X. Dai and M.-A. Elsliger for expert technical assistance, and K. Namba and K. Yonekura for generously sharing coordinates of flagellar filament. X-ray diffraction data sets were collected at SSRL beamline 11-1 and APS beamlines 23ID-B and 23ID-D. Supported by NIH grants AI042266 (I.A.W.), R01 AI080446 (A.V.G.), and RC2 AI087616 (A.V.G.); the Skaggs Institute for Chemical Biology (I.A.W.); and research grants from Cleveland BioLabs Inc. (CBLI) to Roswell Park Cancer Institute (A.V.G.) and Sanford-Burnham Medical Research Institute (A.L.O.). A.L.O. is a paid consultant for and holds stock options in CBLI which has developed the flagellin derivative CBLB502 into a radiation countermeasure. U.S. patent 8,007,812 for pharmacologically optimized FliC derivative CBLB502 is licensed to CBLI by Cleveland Clinic. Reagents are available under a materials transfer agreement. This is Scripps Research Institute manuscript 21369. The data presented in this paper are tabulated in the main paper and the supplementary material. Structure factors and coordinates for TLR5-N12VLR and TLR5-N14VLR/FliC-ΔD0 are deposited in the Protein Data Bank under accession codes 3V44 and 3V47, respectively.
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