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Keywords:

  • evolution;
  • Fn14;
  • NMR;
  • tumor necrosis factor;
  • TWEAK

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

TWEAK (TNF homologue with weak apoptosis-inducing activity) and Fn14 (fibroblast growth factor-inducible protein 14) are members of the tumor necrosis factor (TNF) ligand and receptor super-families. Having observed that Xenopus Fn14 cross-reacts with human TWEAK, despite its relatively low sequence homology to human Fn14, we examined the conservation in tertiary fold and binding interfaces between the two species. Our results, combining NMR solution structure determination, binding assays, extensive site-directed mutagenesis and molecular modeling, reveal that, in addition to the known and previously characterized β−hairpin motif, the helix-loop-helix motif makes an essential contribution to the receptor/ligand binding interface. We further discuss the insight provided by the structural analyses regarding how the cysteine-rich domains of the TNF receptor super-family may have evolved over time.

Database

Structural data are available in the Protein Data Bank/BioMagResBank databases under the accession codes 2KMZ, 2KN0 and 2KN1 and 17237, 17247 and 17252.


Abbreviations
APRIL

a proliferation-inducing ligand

BAFF

B-cell activation factor of the TNF family

BAFFR

BAFF receptor

BCMA

B-cell maturation antigen

CRD

cysteine-rich domain

FACS

fluorescence-activated cell sorting

Fn14

fibroblast growth factor-inducible protein 14

NF-κB

nuclear factor κB

RMSD

root-mean-square deviation

SPR

surface plasmon resonance

TACI

transmembrane activator and calcium signal-modulating cyclophilin ligand interactor

TACI_d2

second CRD of TACI

TNF

tumor necrosis factor

TNFR

tumor necrosis factor receptor

TWEAK

TNF homologue with weak apoptosis inducing activity

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

TNF (tumor necrosis factor) family ligands and their receptors, which include TWEAK (TNF-like weak inducer of apoptosis) and its receptor Fn14 (fibroblast growth factor-inducible immediate-early response protein 14), regulate a wide range of biological processes, including inflammation, lymphocyte survival and activation, as well as tissue repair and remodeling [1, 2]. Although the TWEAK–Fn14 pathway does not seem to play an obligatory role in tissue development and homeostasis, growing evidence indicates that TWEAK-mediated Fn14 activation constitutes an evolutionarily highly conserved [3-5] physiological response to injury by facilitating tissue regeneration and repair [6, 7]. However, dysregulation of the TWEAK–Fn14 pathway under pathological conditions may contribute to amplification of an excessive inflammatory response, pathogenic angiogenesis and tissue remodeling, and inhibition of endogenous repair mechanisms, [8] and has been related to aspects of tumor growth and metastasis [9].

Fn14, together with BCMA (B-cell maturation antigen), BAFFR (B-cell activation factor of the TNF family receptor) and TACI (transmembrane activator and calcium signal-modulating cyclophilin ligand interactor), is one of the smallest tumor necrosis factor (TNF) receptors. It contains just a single extracellular cysteine-rich domain (CRD), and two nested disulfide bridges, a disulfide bond pattern that is different from that of BCMA and TACI [10]. In the present paper, we describe the solution conformations and ligand binding affinities of the soluble ectodomains of human and Xenopus Fn14. Despite considerable divergence in their primary sequences, human and Xenopus Fn14 adopt a nearly identical overall fold, consistent with the observation that both bind to human TWEAK. The presence of conserved structural motifs, coupled with site-directed mutagenesis of both Fn14 and TWEAK, allow us to define the interaction site and to model the interface of the TWEAK–Fn14 complex using the available structures of other TNF receptors (TNFRs) in complex with their ligands [11-13] and the newly determined solution structure of BCMA.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Evolutionary conservation of TWEAK–Fn14 interactions

Human and Xenopus Fn14 (referred to as hFn14 and xeFn14) share only 37% sequence identity overall and 40% in the extracellular domain [14], but share several common features (Fig. 1), including a single extracellular CRD with six identically spaced cysteine residues that are separated by short stretches of homologous amino acids. To test the role of the conserved residues in interacting with TWEAK, we measured the binding of human or Xenopus Fn14 to human TWEAK by fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Figure 2A shows a FACS analysis in which we transfected full-length hFn14 or xeFn14 into 293T cells and measured the binding of Fc-hTWEAK (a fusion protein between the Fc portion of human IgG1 and the TNF homology domain of hTWEAK). Similarly, Fig. 2B shows ELISA results for Fc-hTWEAK titrated on plates coated with human or Xenopus Fn14. We observed comparable binding to proteins of both species, with an EC50 of 0.3 nm for Fc-hTWEAK binding to each receptor. The values are in agreement with those recently reported for binding of a conjugated form of human TWEAK to hFn14 in human HT1080 and HT29 cells [15]. The high affinity detected for the xeFn14/hTWEAK interaction prompted us to measure the true, monovalent affinity of human and Xenopus Fn14 for human TWEAK by SPR. Figure 2C,D shows the results of equilibrium binding of monomeric Xenopus or human Fn14 to hTWEAK measured by SPR. hFn14 binds to hTWEAK with a KD of 65 nm (Fig. 2C), while xeFn14 binds to hTWEAK with a KD of 1.3 μm (Fig. 2D); a 20-fold lower affinity than hFn14. This difference, which cannot be detected in avidity-prone, multivalent interaction assay formats [16], becomes apparent only when the single-site binding affinity is measured. Importantly, hTWEAK induced similarly robust nuclear factor κB (NF-κB) activation when co-transfected with either hFn14 or xeFn14 in 293T cells, indicating that the observed binding interaction with xeFn14 also results in functional intracellular signaling (Fig. 2E). By comparison, cells transfected with TWEAK alone show only a slight increase in NF-κB signaling, which was negligible compared to that obtained by co-transfection with Fn14 (1.5-fold increase, = 0.01, which may be attributed to stimulation of endogenous Fn14). Taken together, these data strongly suggest that endogenous interactions between TWEAK and Fn14 are mostly highly conserved during evolution.

image

Figure 1. Sequence alignment of Fn14 and other small TNF receptors. (A) Sequence alignment of Fn14 from human (Homo sapiens), mouse (Mus musculus), frog (Xenopus laevis), fish (Gasterosteus aculeatus) and a protochordate (Branchiostoma floridae). Conserved residues in the extracellular domain are shaded. Numbering corresponds to the human sequence. TMD, transmembrane domain; TNF receptor associated factor (TRAF), consensus TRAF binding site. Exon boundaries, when known, are indicated. (B) Sequence alignment of the central portion of the extracellular domains of single-CRD TNF receptors and TNFR1. The elements of secondary structure (β-sheets in green, α-helices in red) are indicated, as well as the disulfide bonds. Residues with a purple background were experimentally shown to participate in ligand binding (for Fn14, BCMA, TACI and BAFFR) or to contact the ligand in the ligand–receptor complex (TNFR1). Yellow and orange backgrounds indicate the Cys pairs. The module composition of the CRD is shown on the right. dWengen, Drosophila Wengen.

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image

Figure 2. Cross-species binding of Fn14 to human TWEAK. (A) 293T cells were transiently transfected with the indicated amount of plasmids encoding full-length hFn14 or xeFn14, stained with Fc-hTWEAK at the indicated concentrations, and analyzed by FACS. MFI, mean fluorescence intensity. (B) ELISA assay. Immobilized hFn14 and xeFn14 binding to Fc-hTWEAK and detection with secondary reagents against human Fc. A four-parameter equation fitting yielded an apparent EC50 of 0.3 nm. (C,D) Binding of monomeric Fn14 (in solution) to immobilized trimeric His-tagged hTWEAK on a Biacore chip. The plot of the signal achieved at equilibrium (Req) as a function of the hFn14 (C) or xeFn14 (D) concentration was fitted to a single-site binding model as described in Experimental procedures. (E) Induction of NF-κB-luciferase reporter activity in 293T cells transfected with varying amounts of xeFn14 or hFn14 plasmids, in the presence or absence of a fixed amount of co-transfected TWEAK. The signal in cells expressing TWEAK in the absence of co-transfected Fn14 is represented by the white square and the white circle (hidden below the white square) at 0 ng per well of Fn14. NF-κB activity is expressed as a fold increase relative to cells transfected without Fn14.

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Solution structure of hFn14 and xeFn14 extracellular domains

To understand the structural basis underlying the high level of cross-species conservation of TWEAK–Fn14 interactions, we determined the structure of the extracellular ligand-binding domain of both hFn14 and xeFn14 by NMR (Fig. 3A,B; Table 1). The solution structure obtained for hFn14 is in agreement with the published structure [17], with pairwise root-mean-square deviations (RMSDs) in the structured regions of 0.62 and 0.47 Å (family of 20 structures versus PDB code 2RPJ, structure 1) and the same tertiary arrangement. hFn14 and xeFn14 share strong similarity in terms of the NMR spectral pattern (chemical shifts and Nuclear Overhauser Effects (NOEs), Fig. S1), which translates to highly similar secondary and tertiary structures (RMSD 1.0 Å over secondary structure regions: residues 40–52 and 61–64, backbone atoms, Fig. 3B; numbering as in hFn14). The disulfide pattern of hFn14 was determined using tandem mass spectrometry [10], and was further confirmed by NMR for both hFn14 and xeFn14. Both proteins adopt a nested pattern for the two most C-terminal disulfide bonds, in contrast to the intercalated pattern observed in BCMA and TACI_d2 (second CRD of TACI), the two TNFR super-family members previously thought to be most closely related to Fn14 (Figs 1B and 3A,C). The structures of the highly ordered β-hairpin domains (RMSDs 0.24 and 0.20 Å, respectively, for each ensemble of 20 structures of hFn14 and xeFn14) converge to an RMSD of 0.56 Å. This area of the molecule has been previously reported to provide the majority of the interaction interface with the ligand for BCMA and BAFFR, and to be essential for binding in TACI and multi-domain TNF receptors (TNFR1 and DR5, death receptor 5) [18, 19].

image

Figure 3. NMR solution structure of human and Xenopus Fn14. (A) Superposition of 20 low-energy NMR-derived structures of hFn14 (backbone atoms 36–67). The three disulfide bridges are shown by yellow ball-and-stick models for one of the structures. The boundaries of the A1 and C2 structural modules are indicated by arrows. (B) Superposition of low-energy structures of hFn14 (gray) and xeFn14 (red), displayed as ribbons. Residues 36, 40–52 and 61–64 of hFn14 and corresponding residues of xeFn14 were used in the superposition. (C) Top view of hFn14 with cysteines highlighted by yellow ball-and-stick models. The arrows indicate the changes in disulfide connectivity that are required to swap from nested to intercalated disulfide bridges in the C2 module. (D) The hydrophobic core of hFn14 comprises Phe63 and Met50 (carbon atoms in green), and is surrounded by small amino acids (Ser41, Ser43, Ser54 and Ser61, carbon atoms in blue). Protons are colored white, oxygens are colored red, and sulfur is colored yellow.

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Table 1. Restraints and statistics for the ensemble of 20 structures of hFn14, xeFn14 and BCMA
 hFn14xeFn14BCMA
  1. a

    vadar was used to assess the stereochemical quality of the structures.

NOE restraints230218279
Intra-residue795678
Sequential8981100
Medium range 1 < |i − j | < 5283145
Long range |i − j | ≥ 5345056
RMSD from experiment
NOE constraints (Å)0.0150.0350.060
RMSD from idealized geometry
Bonds (Å)0.0020.0030.005
Angles (°)0.30.40.7
Ramachandran analysisa
Most favored regions (%)56.3 ± 6.761.2 ± 5.056.0 ± 4.5
Additionally allowed regions (%)34.2 ± 5.734.0 ± 5.930.8 ± 4.5
Generously allowed regions (%)5.8 ± 3.43.1 ± 2.38.9 ± 2.4
Disallowed regions (%)0.2 ± 0.70.1 ± 0.40.2 ± 0.6
Average pairwise RMSDs (Å)
Ser40/xPro31 to Cys67/xCys580.7470.760 

The relative arrangement of the C-terminal lobe of Fn14 proteins (helix 1, residues 52–55, loop, and helix 2, residues 62–69) and the N-terminal β-hairpin is well defined by a number of long-range NOEs radiating from the central aromatic residue Phe63/xPhe54 (for simplicity, the first residue refers to hFn14 and the residue prefixed ‘x’ is the corresponding residue in xeFn14; NOEs to Ser41/xAla32, Trp42/xTyr33, Ser43/xSer34, Met50/xMet41). In the case of xeFn14, long-range contacts between the two acidic residues xAsp53 and xAsp36 may also be clearly observed. To quantitatively evaluate NOEs and their impact on the final structures, we utilized the program queen [20]. The average information, Iave, for long-range restraints connecting residues in the loop (Phe63, Gly66, Cys67; xPhe54, xAsn57 and xCys58) to residues in the N-terminal region (Ser41, Ser43 and xArg31–xSer34) is 32% and 35%, respectively, of the total structural information, indicating a dominant role in the structure. NOEs in the helix 2 region have a higher contribution to the dataset for hFn14 than for xeFn14 (i.e. a better chemical shift dispersion is observed). Correspondingly, helix 2 in the human protein is better defined and folds into a more compact coil (Fig. 3B).

The observed structures closely resemble that of TACI_d2 (RMSD 0.52 Å between residues 40 and 50 of hFn14, and residues 77 and 87 of TACI_d2, lowest NOE violation structure, PDB code 1XUT) (Fig. 4A,C).

image

Figure 4. Comparison of the structural features of hFn14 with BCMA, TACI_d2 and BAFFR. Selected residues are displayed by ball-and-stick models: green, residues in the hydrophobic core; dark blue, residues relevant for binding in the β-hairpin and helix 2. The conserved aromatic residue is shown in light blue and disulfide bridges are shown in yellow. (A) NMR structure of hFn14. (B) NMR structure of human BCMA determined in this study. The Tyr13 that fulfils a similar structural role to Phe63 of Fn14 is shown. (C) NMR structure of hTACI_d2 (PDB code 1XUT). (D) Crystal structure of hBAFFR (PDB code 1OQE). The structures shown in (A–D) were first superimposed on their β-hairpins. Note that the N- and C-terminal domains adopt different relative orientations in Fn14 than in BCMA and TACI_d2, which may allow engagement of different epitopes in ligand binding.

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Solution structure of BCMA

The relative orientation of the N- and C-terminal lobes of Fn14 determined by NMR is strikingly different from those of TACI_d2 and especially BCMA as determined by crystallography (Fig. 4) [12, 13]. We confirmed that the solution structure of BCMA is highly similar to the crystal structures of BCMA determined previously in complex with BAFF (B-cell activation factor of the TNF family) or APRIL (a proliferation-inducing ligand) [12, 21]; the backbone RMSD over the ordered regions is 1.06 Å (lowest NOE violation structure versus PDB code 1OQD). A similar analysis of the NMR restraints (queen) indicates that the relative orientation of the N- and C-terminal lobes is well characterized by long-range NOEs, which constitute about 19% of the total structural information (sum of Iave), involving residues homologous to those observed for Fn14 (Pro34, Thr36, Tyr40, Cys41 to Asn11, Tyr13 and Phe14).

Tertiary fold and binding epitopes for single-CRD TNFRs

Overall, the fold of Fn14 is similar to those of BCMA and TACI_d2 and partially similar to that of BAFFR, with the most striking similarity in the β-hairpin. The conserved DXL motif, located at the tip of the β-hairpin and essential for ligand binding [11, 12], is replaced in Fn14 by an SXDL sequence that contributes to ligand binding. Whereas the hydrophobic core in h/xeFn14 is very compact (Phe63/xPhe54 and Met50/xMet41, surrounded by a ring of small amino acids: Ser41/xAla32, Ser43/xSer34, Ser54/xVal45 and Ser61/xSer52, Fig. 3D), the hydrophobic core of TACI_d2 involves a number of bulkier hydrophobic residues, including Phe78, Ile87, Ile92 and Pro97 [12] (Fig. 4C), causing a tertiary arrangement that is progressively more open going from Fn14 to TACI_d2 to BCMA (the N- and C-terminal lobes arrange in an ‘open clamp’ versus ‘closed clamp’ in Fn14, Fig. 4).

As a functional consequence of the tertiary structure, BCMA and BAFFR engage BAFF (and APRIL) mostly through the β-hairpin interface [13, 21], with no fundamental contribution from residues in the C-terminal helix-loop-helix region. The intermediate conformation in TACI_d2 involves a wider binding surface to APRIL, encompassing the concave face of the molecule (DXL hairpin plus residues in the helix-loop-helix motif) [12]. In the ‘closed clamp’ conformation seen in both human and xeFn14, the β−hairpin and the helix-loop-helix motifs are in close proximity and define the binding epitope presented to TWEAK (Fig. 4).

A discontinuous binding epitope: mutagenesis and molecular structure

Previous studies have concluded that minimal ligand-induced structural changes occur when BAFFR, BCMA or TACI_d2 bind their respective ligands [11-13], an observation confirmed by the present solution structure of BCMA. A model of hFn14 bound to TWEAK was constructed based on the assumptions that (a) the structure of free Fn14 and TWEAK-bound Fn14 is similar, and (b) the region contacted by Fn14 in TWEAK corresponds to that contacted by BAFFR, BCMA and TACI_d2 in BAFF and APRIL. We based our homology model on the available structure of the APRIL–TACI complex (PDB code 1XU1) [12]. Fn14 contacts TWEAK along a relatively narrow but elongated surface comprising, on one hand, the C-terminal end of the CD loop plus β-strand E of TWEAK, and, on the other hand, the β−hairpin and part of the C-terminal portion of Fn14.

The molecular model of the TWEAK–Fn14 complex, which takes into account the experimentally determined structure of Fn14, clearly highlights that residues belonging to the two lobes of Fn14 (the β−hairpin and the helix-loop-helix domain) make determinant contributions to the binding interface. Based on the complex structure, selected point mutations were introduced in Fn14 and TWEAK in order to probe potential residues involved in the receptor–ligand interactions. Full-length forms of Fn14 were expressed in 293T cells and stained with FLAG-tagged TWEAK in FACS analysis, or with a monoclonal antibody against Fn14 (ITEM-4) to control expression levels (Fig. 5A). Leu46 was deemed important for binding, and its mutation (L46A) reduced the binding affinity to TWEAK; all other Fn14 mutants tested bound FLAG-tagged TWEAK as efficiently as wild-type Fn14 did (Fig. 5B). Consequently, we introduced a series of double mutations in the hFn14 sequence that combine a residue from the β-hairpin's tip and a residue from the helix-loop-helix, expressed them in 293T cells, and tested them for binding to hTWEAK. Several double mutants resulted in partial or complete loss of binding to TWEAK: L46A/P59A, L46A/S61A, L46A/D62A and L46A/F63A (Fig. 5C). To confirm the importance of the SXDL motif, a number of double mutants with two mutated residues within the β-hairpin were also examined. Mutants S43A/L46A, D45A/L46A, D47A/L46A and K48A/L46A almost completely lost the ability to bind to TWEAK (Fig. 5C).

image

Figure 5. Mutagenesis of Fn14 and TWEAK. (A) The indicated full-length forms of wild-type or mutant Fn14 were expressed in 293T cells and stained by FACS with FLAG-tagged TWEAK at the indicated concentrations (1 μg·mL−1 corresponds to 50 nm), or with a monoclonal antibody against Fn14 (ITEM-4) as an expression control. (B) Binding of wild-type TWEAK to wild-type and mutant Fn14 was monitored as shown in (A). The mean fluorescence intensity of TWEAK binding on cells expressing 102–103 fluorescence units of EGFP [rectangle shown in the first scattergram of (A)] was normalized to the staining intensity obtained with ITEM-4, and plotted as a function of TWEAK concentration. (C) As for (B), except that the Fn14 mutations were combined with the L46A mutation. (D) 293T cells transfected with full-length wild-type Fn14 were stained with the indicated concentration of FLAG-tagged TWEAK with the indicated mutations. Only TWEAK mutation Y176A abolished binding to Fn14. (E) As for (D), but with the Fn14 L46A mutant. The TWEAK mutants K178A, R190A and L192E all failed to interact with the Fn14 L46A mutant, which displays a weaker affinity for TWEAK.

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Similarly, we mapped the TWEAK binding interface, revealing the importance of Tyr176 (Y176A completely abolishes binding; Fig. 5D), and the contribution of Lys178, Arg190 and Leu198 (K178A, R190A and L192E all failed to interact with Fn14 L46A, the weakened receptor for TWEAK; Fig. 5E). The residues thus discovered all appear to be involved in specific receptor–ligand interactions, and are highlighted in Fig. 6. For example, Leu46 on Fn14 interacts with Leu192 of TWEAK, Asp47 of Fn14 interacts with Lys235 of TWEAK, and Ser61/Asp62 of Fn14 interact with Lys178 of TWEAK. Pro59, which has a moderate effect on binding affinity in double mutant Fn14, may play a structural role or additionally directly participate in binding, similar to the corresponding residue Pro97 of TACI_d2 (part of the hydrophobic surface that interacts with APRIL). We also attribute a structural role to Met50: as part of the hydrophobic core of Fn14 (only 20% of its surface is accessible), it is not predicted to contact TWEAK directly but rather to play a major role in determining the relative orientation of the N- and C-terminal lobes. A similar effect was reported previously for the equivalent position in TACI_d2 (Ile87) [12] (Fig. 4C). Several residues showed no contribution to binding (Fig. 5C): Arg56, Arg58 and Leu65 point away from the TWEAK interface in the model and make no or little contact with TWEAK, while His60 points toward Glu194 and Phe195 of TWEAK.

image

Figure 6. Molecular model of the TWEAK–Fn14 complex. (A) Residues of hFn14 that were tested in the mutagenesis study. Residues shown in red were critical for ligand binding (in combination with mutation L46A), the residue shown in orange decreased binding, and residues shown in green had no or little effect on ligand binding when mutated to alanine. Residues marked with an asterisk are within 4 Å of TWEAK in the model. Note that residues that are important for binding are located in both the A1 and C2 modules. (B) Model of hFn14 binding to hTWEAK. Residues of both Fn14 and TWEAK that are likely to participate directly in the interaction and whose mutation affects the binding are highlighted (green, hydrophobic; red, acidic; blue, basic). The TWEAK trimer is represented as ribbons in various shades of yellow. Fn14 is represented as a gray ribbon. Fn14 in (A) and (B) is rotated ~ 180°, to allow optimal viewing of the residues described.

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Within the series of single-mutant hFn14 constructs, the finding that each mutation alone did not disrupt ligand binding (or only reduced it, e.g. L46A) indicates that none of these mutations resulted in gross unfolding or intracellular retention of hFn14. It is noteworthy that our results are in contrast with a previous report indicating that the D62A mutation totally prevented the secretion of Fn14 protein, and several single point mutations disrupted binding [14]. Secretion of the soluble D62A mutant may be affected by the use of full-length human Fn14 in this study, versus use of a murine sequence of the extracellular domain of Fn14 fused to murine Fc and a Myc tag [14]. We also tested mutations in neighboring amino acids, S61A and F63A, which yielded Fn14 mutants that are able to bind TWEAK. In addition, the binding assay modalities were largely different: binding affinities in this study were compared by FACS analysis on cells expressing the described Fn14 mutants with increasing concentrations of FLAG-tagged TWEAK (> 10 μg·mL−1), whereas the previous study [14] measured binding affinity to TWEAK immobilized on a solid surface at two concentrations of purified receptors, and the weakest detected binding was reported with a KD of 71 nm. To confirm correct folding of the mutant proteins, a representative double mutant, L46A/R58A hFn14, was expressed and purified, and examined by 1H,15N-HSQC NMR. The protein spectrum (Fig. S2) closely tracks that of wild-type hFn14, with chemical shift perturbations corresponding only to the sites adjacent to the mutated residues as expected, indicating that the wild-type and mutant proteins are similarly folded.

Taken together, our data indicate that the Fn14–TWEAK interaction involves elements from the β-hairpin as well as from the helix-loop-helix motif. The observation that single point mutations are well tolerated appears to indicate that the multiple sites of this non-contiguous interaction interface may contribute substantially to the energy of binding, which should be taken into account for development of low-molecular-weight molecules intended to modulate the TWEAK–Fn14 interaction.

Fn14: the evolutionary perspective

The TNF super-families of ligands and receptors have expanded considerably during evolution, from a single pair in Drosophila (Eiger–Wengen) to more than 20 in human, as higher organisms came to rely more and more on these ligand–receptor interactions for controlling cell fate decisions. However, the evolutionary origin and diversification of these pairs is incompletely understood. In the present study, we provide experimental evidence indicating that the interface between the TNF ligand TWEAK and its cognate receptor Fn14 has been greatly conserved during evolution.

Although it may seem remarkable that Xenopus Fn14 cross-reacts with human TWEAK, inter-species cross-reactivity is not uncommon in the TNF family: 72% of TNF ligands and receptors cross-react between human and mouse [22], human BAFF binds to chicken BAFFR [23], chicken BAFF binds to human BCMA [24], and mammalian ectodysplasin-A (EDA) is even active in fishes [25]. The strong structural similarity observed between human and Xenopus Fn14 despite divergent primary sequences is probably due to the presence of a structural core stabilized by disulfide bonds, as shown previously for other protein families [26, 27].

Most TNF receptor family members, such as TNFR1, TNFR2 and Fas, contain three or four cysteine-rich domains, usually composed of A and B modules (see [2] for classification), suggesting that these receptors may have arisen by exon duplication of an ancestral cysteine-rich domain such as that found in Wengen in Drosophila [28] (Fig. 1B). The relatively recent identification of single-CRD receptors in mammals raised the intriguing possibility that they may represent ancestral molecules from which multi-CRD mammalian TNF receptors evolved [12]. The TWEAK–Fn14 pathway may be one of the most ‘ancient’ from the perspective of evolution. The other two single-CRD TNF receptors BAFFR and BCMA regulate B-cell function in vertebrates only, whereas an Fn14-like sequence with perfectly conserved inter-cysteine spacing and exon–intron boundaries is found in the protochordate Branchiostoma floridae (Fig. 1A). This suggests that the origin of the TWEAK–Fn14 pathway preceded vertebrate evolution and the development of adaptive immunity.

The structure of Fn14 allows formulation of a hypothesis regarding the evolutionary relationship between modules that differ in their patterns of disulfide bridges. Although the A1–C2 cysteine-bridging pattern described in both human and Xenopus Fn14 (the two C2 disulfides are nested) contrasts with the A1–D2 pattern of BCMA and TACI (intercalated disulfides), the three-dimensional structure of Fn14 is able to accommodate the A1–D2 disulfide pattern with only minor structural adjustments (Fig. 3C), suggesting that a certain level of ‘ambiguity’ in disulfide linkage formation may have existed in the primordial single-CRD TNF receptor, and that this flexibility may account for the emergence of various modules found in mammalian TNF receptors. Similarly, swapping of disulfide pairing has been reported to produce bioactive disulfide bond isomers of epidermal growth factor-like domains [29].

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Protein preparation

Soluble FLAG-tagged human TWEAK or Fc-hTWEAK (residues 106–249; a fusion protein between the Fc portion of human IgG1 and the TNF homology domain of hTWEAK) were generated and characterized as previously described [22]. Full-length, untagged human Fn14 was cloned into the mammalian expression vector PCR3 (Life Technologies, Grand Island, NY, USA). Point mutations were introduced by conventional PCR-based methods in full-length Fn14 and hTWEAK, and the resulting plasmids were sequenced on both strands. His-tagged hTWEAK (residues 106–249) and the soluble ectodomains of human Fn14 (residues 28–80) and Xenopus Fn14 (residues 23–72) fused to Myc and His tags were cloned and expressed in Pichia pastoris as previously described [30]. The molecular weight of hFn14 as determined by MS spectrometry was 7617 Da, corresponding to the oxidized state for all six cysteine residues with no post-translational modification [10]. Uniformly 15N-labeled hFn14 and xeFn14 for NMR studies were prepared from cultures supplemented with (15NH4)2SO4 as previously described [31]. Additional forms of soluble Fn14 used in this study included hFn14-Fc and xeFn14-Fc (CHO or 293T mammalian cells). The L46A/R58A mutant of hFn14 was initially cloned in a pQE vector (Qiagen, Germantown, MD, USA) and subjected to PCR-based site-directed mutagenesis to introduce A46 and A58. It was subsequently sub-cloned in a modified pET32b vector (Novagen/EMD Millipore, Billerica, MA, USA) between the BamHI and XhoI restriction sites, as a His6-tagged thioredoxin fusion protein containing a tobacco etch virus (TEV) cleavage site, and expressed and purified from Escherichia coli BL21 Star (DE3) (Life Technologies) as previously described [32]. The last purification step of the cleaved L46A/R58A hFn14 mutant consisted of size-exclusion chromatography using Superdex Peptide 10/300GL (GE Healthcare, Pittsburgh, PA), in which the protein eluted at the expected mass for a monomeric species. The protein was characterized by SDS/PAGE and 1H,15N NMR spectroscopy.

The cysteine-rich domain of human BCMA (residues 2–50) was synthesized by solid-phase peptide synthesis (Methods S1).

ELISA assay

Microtiter plates were coated with hFn14 or xeFn14 at 10 μg·mL−1 in 100 mm sodium carbonate buffer, pH 9.2, and then blocked with ELISA buffer (10 mm phosphate, 137 mm NaCl, 2.7 mm KCl, pH 7.4, plus 5% non-fat dry milk, 0.05% Tween-20). Plates were washed at room temperature twice (ELISA plate washer) with wash buffer (10 mm phosphate, 137 mm NaCl, 2.7 mm KCl, pH 7.4, 0.05% Tween-20) and the indicated concentrations of Fc-hTWEAK in ELISA buffer were added for 1 h at room temperature. Plates were washed as above, and bound Fc-hTWEAK was detected by adding horseradish peroxidase-labeled goat IgG against human Fc (Fc-specific) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) at a dilution of 1:1000 in ELISA buffer for 1 h at room temperature. Plates were developed using TMB substrate (Pierce, Rockford, IL, USA), the reaction was stopped by addition of 2 M H2SO4, and absorbance was read at 450 nm. EC50 values were determined from an empirical four-parameter fit to the data:

  • display math

where a is the upper asymptote, b is the mid-point (EC50), c is the slope of the linear part of the curve, and d is the lower asymptote.

Cell-surface binding analysis

293T cells were transiently transfected by the calcium phosphate precipitation method with plasmids containing full-length wild-type human or xeFn14 cDNAs, together with an enhanced green fluorescent protein (EGFP) expression plasmid [33]. Cells were harvested 24 h post-transfection and stained with varying amounts of Fc-hTWEAK, followed by fluorescein isothiocyanate- or R-Phycoerythrin (PE)-conjugated goat secondary antibodies against human Fc (#2040-09, Southern Biotech, Birmingham, AL, USA). This allowed semi-quantitative estimation of the apparent KD values for binding between Fc-hTWEAK and hFn14 or xeFn14 expressed on the cell surface. Alternatively, 293T cells were co-transfected by the calcium phosphate method with plasmids encoding EGFP and human Fn14 (wild-type, or with the indicated single or double mutations). Cells were stained 48 h post-transfection with 50 μL FLAG-tagged hTWEAK (wild-type or mutants) at the indicated concentrations, followed by biotinylated ANTI-FLAG M2 antibody (Sigma-Aldrich, St Louis, MO, USA) and PE-coupled streptavidin. Cells were also stained using biotinylated ITEM-4 (monoclonal antibody against Fn14), followed by PE-coupled streptavidin. The mean fluorescence intensity of FLAG-tagged hTWEAK staining was measured on cells expressing intermediate levels of EGFP (fluorescence intensity 100–1000), and was normalized to the level of Fn14 expression measured using ITEM-4.

Surface plasmon resonance assay

All experiments were performed using a Biacore 3000 instrument (GE Healthcare). His-tagged hTWEAK [16] was immobilized on a CM5 sensorchip as described previously [34]. Soluble monomeric human or Xenopus Fn14 was diluted in Biacore assay buffer (Biacore buffer + 0.05% BSA) to the indicated concentrations, and injected over the TWEAK-derivatized or control surface at a flow rate of 50 μL·min−1. The TWEAK surface was regenerated by two 30 s injections of 10 mm H3PO4. In all cases, binding to the underivatized chip was negligible. Affinity was determined by fitting plots of the response value where binding has come to equilibrium (Req) versus concentration to a hyperbolic single-site binding equation [28].

NF-κB luciferase assay

293T cells were plated at 104 cells per well in 96-well plates in 100 μL Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Two days later, plasmid mixes were prepared in one volume of Dulbecco's modified Eagle's medium without supplement, mixed with 0.07 volumes of Polyfect (Qiagen) for 5 min, diluted with five volumes of Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 25 μL were distributed on cells in 50 μl fresh complete medium. Each well received hFn14 or xeFn14 plasmids (20 ng, threefold dilutions), with or without 4 ng of hTWEAK plasmid and with 7.5 ng each of NF-κB firefly luciferase, renilla luciferase and EGFP plasmids, plus empty vector to achieve a total of 70 ng of plasmid per well. After 48 h, cells were washed, lysed and assayed for firefly and renilla luciferase using a dual reporter luciferase assay (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to that of renilla luciferase. NF-κB activation was expressed as the fold increase relative to signals obtained in cells transfected without Fn14.

NMR spectroscopy

NMR samples typically consisted of 500–700 μm 15N-xeFn14, 15N-hFn14 or hFn14 in 10 mm phosphate, 137 mm NaCl, 2.7 mm KCl (pH 7.4) and 5% D2O. BCMA (900 μm) was dissolved in 10 mm phosphate buffer, pH 6.0, 0.02% NaN3, 5% D2O. NMR spectra were acquired on a Bruker (Bruker BioSpin Corporation, Billerica, MA, USA) Avance 600 spectrometer equipped with a triple resonance CryoProbe or TXI probe, at 10–45 °C. 1H and 15N assignments were obtained using three-dimensional 15N-edited NOESY-HSQC (100 ms) and TOCSY-HSQC experiments [35] (64, 200 and 1024 complex points in the 15N and 1H dimensions) and two-dimensional homonuclear experiments: TOCSY, NOESY (90 ms) and double quantum-filtered COSY [36]. All NMR data were processed using TOPSPIN (version 1.3; Bruker) and analyzed using Sparky [37].

Structure calculation

Complete proton and nitrogen resonances assignment was achieved as described previously [38]. Distance restraints were derived from the 3D 1H-15N edited NOESY and 2D 1H-1H NOESY experiments, with mixing times varying between 80 and 100 ms. For each protein, 100 structures were calculated using the torsion angle simulated annealing protocol of CNX (version 2002; Accelrys Inc., San Diego, CA, USA) using standard parameters [39], and the 20 structures with lowest total energy were chosen for analysis. The residues in the Myc/His tag were excluded from the structure calculation. The quality of the structures was analyzed using vadar [40], and the experimental NMR restraints were quantitatively evaluated using queen [20] (calculation of the unique information, Iuni, and average information content sampled throughout the complete dataset, Iave, over 25 iterations, for each restraint). The structures were visualized using Chimera [41].

Disulfide bond determination

Disulfide bond assignments for all three structures were confirmed by NMR utilizing the method described by Klaus et al. [42], which determines the most likely pattern of disulfide bonds based on calculation of all possible distances between the Cβ of cysteine residues involved in disulfide bonds within the initial family of NMR structures (calculated in the absence of explicit S–S bond definitions). To investigate the consequences of disulfide swapping, a set of structures with the alternative disulfide configuration was calculated as well.

Molecular modeling of TWEAK–Fn14 interactions

The model of TWEAK was generated by template forcing homologous sequence regions (as generated by blast [43]) to the three-dimensional structure of APRIL (PDB entry 1XU1) [12] using InsightII (Accelrys). The model of the TWEAK trimer and the structure of Fn14 were superimposed on the crystal structure of APRIL and the bound second CRD domain of TACI (TACI_d2) (PDB entry 1XU1) [12], respectively, using InsightII. The complex was then subjected to extensive energy minimization (steepest descent followed by the quasi-Newton-Raphson method VA09a, until the derivatives were < 0.5 kcal·mol−1·Å−1, CVFF91 forcefield), first with the NMR distance constraints applied to maintain the Fn14 structure (force constant of 100 kcal·mol−1·Å−2), and then with no constraints, allowing the complex to relax.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported in part by grants from the Swiss National Science Foundation (to P.S.). We thank Hideo Yagita (Juntendo University, Department of Immunology, Tokyo, Japan) for the gift of ITEM-4 antibody against Fn14. M.P. wishes to thank Jared Cochran (Indiana University, Molecular and Cellular Biochemistry Department) for the gift of the modified pET32 plasmid, and Dale Mierke (Dartmouth College) for assistance in the molecular modeling efforts.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
febs12206-sup-0001-FigS1-S2.zipapplication/ZIP558K

Fig. S1. 2D NOESY spectra of hFn14 and xeFn14.

Fig. S2. 1H,15N HSQC spectra of hFn14 and the L46A/R58A hFn14 mutant.

Methods S1. Methods for the synthesis of BCMA.

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