Solution structure of the cysteine-rich domain in Fn14, a member of the tumor necrosis factor receptor superfamily

Abstract

Fn14 is the smallest member of the tumor necrosis factor (TNF) receptor superfamily, and specifically binds to its ligand, TWEAK (TNF-like weak inducer of apoptosis), which is a member of the TNF superfamily. The receptor-ligand recognition between Fn14 and TWEAK induces a variety of cellular processes for tissue remodeling and is also involved in the pathogenesis of some human diseases, such as cancer, chronic autoimmune diseases, and acute ischaemic stroke. The extracellular ligand-binding region of Fn14 is composed of 53 amino acid residues and forms a single, cysteine-rich domain (CRD). In this study, we determined the solution structure of the Fn14 CRD (Glu28-Ala70) by heteronuclear NMR, with a (13)C-/(15)N-labeled sample. The tertiary structure of the CRD comprises a beta-sheet with two strands, followed by a 3(10) helix and a C-terminal alpha-helix, and is stabilized by three disulfide bonds connecting Cys36-Cys49, Cys52-Cys67, and Cys55-Cys64. Comparison of the disulfide bond connectivities and the tertiary structures with those of other CRDs revealed that the Fn14 CRD is similar to the fourth CRD of TNF receptor 1 (A1-C2 module type), but not to the CRD of B-cell maturation antigen and the second CRD of transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor (A1-D2 module type). This is the first structural report about the A1-C2 type CRD that could bind to the known target.

Figure 1

Structure-based multiple sequence alignment of the CRDs of the A1-C2 and A1-D2 modules of the TNFRSF members. Cys residues are highlighted in red and their disulfide bond connectivities are shown above the sequence alignment. Labels and elements of the secondary structure of the Fn14 CRD are reported above the aligned sequences. The crucial residues reported by mutagenesis experiments are marked by dots above the aligned sequence. The sequence alignment output was made with the ESPript program.

Figure 2

Solution structure of the CRD of human Fn14. (A) Stereo-view of the best 20 structures of the CRD (residues Glu28-Ala70), calculated by CYANA2.1. The helices, the β-sheet, and the loop regions are shown in red, cyan, and gray, respectively. (B) Ribbon presentation of the lowest energy structure of the Fn14 CRD (core part: residues Ala34- Ala69). The colors for the CRD are the same as in A. The side chains of the disulfide bonds are shown in yellow. (C) Electrostatic surface presentation of the CRD domain. The blue and red colors represent positive and negative electrostatic surface potential, respectively. (D) Hydrophobic surface presentation of the CRD domain. The ribbon diagram, electrostatic potential, and hydrophobic surface presentations have the same orientation.

Figure 3

Comparison of the A1-C2 and A1-D2 module structures. (A) Ribbon diagram of the A1-C2 module structure of the Fn14 CRD, with disulfide bonds in yellow. (B) Ribbon diagram of the A1-C2 module structure of the TNFR1-d4, with disulfide bonds in yellow. In A and B, the A1 and C2 modules are shown in cyan and red, respectively. (C) Ribbon diagram of the A1-D2 module structure of the BCMA CRD, with disulfide bonds in yellow. The A1 and C2 modules are colored cyan and magenta, respectively. In A, B, and C, the change of the C-terminal α-helix location between the A1-C2 and A1-D2 module structures approximately corresponds to a 60° rotation, and the orientations of Fn14 CRD, TNFR1-d4, and BCMA-d are the same as those in the ribbon diagram on the right in Figure 2(B). (D) Superposition of the ribbon structures in A, B, and C. (E, F, and G) Ribbon diagrams with selected side chains of the CRD module structures of Fn14 (E), TNFR1 (F), and BCMA (G). The positively charged, negatively charged, hydrophobic, and other residues are colored blue, red, green, and magenta, respectively. The crucial residues (D45, K48, M50, and D62) in the human Fn14 CRD, identified by the mutagenesis study, are shown with red annotations. (H) Superposition of the structures in E, F, and G. Backbones are depicted by black lines, and side chains are shown by stick models, colored as in E, F, and G. In (E–H), the orientations correspond to a −120° rotation around the x axis from the right ribbon diagram in Figure 2(B).