Structures of APRIL-Receptor Complexes: LIKE BCMA, TACI EMPLOYS ONLY A SINGLE CYSTEINE-RICH DOMAIN FOR HIGH AFFINITY LIGAND BINDING

Sarah G Hymowitz,Darshana R Patel,Heidi J.A Wallweber,Steven Runyon,Minhong Yan,Jianping Yin,Stephanie K Shriver,Nathaniel C Gordon,Borlan Pan,Nicholas J Skelton,Robert F Kelley,Melissa A Starovasnik

doi:10.1074/jbc.m411714200

Abstract

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.

Highlights

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function
We report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with cocrystal structures of APRIL1⁄7TACI_d2 and APRIL1⁄7BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single cysteine-rich domains (CRDs), and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system
The Membrane-proximal CRD Is the High Affinity Ligand Binding Domain of TACI—We have shown that the second CRD of TACI (TACI_d2) binds both APRIL and BAFF with high affinity, whereas the first CRD does not

Summary

EXPERIMENTAL PROCEDURES

Protein Production—Murine APRIL was produced as described previously [28]. Murine APRIL was used throughout the study because it is better behaved biochemically than recombinant human APRIL. For production of human TACI_d2, DNA encoding TACI residues 68 –109 was subcloned into the pET32a expression vector (Novagen), creating a fusion protein consisting of an N-terminal thioredoxin domain followed by a His tag, thrombin cleavage site, TACI_d2. Libraries were sorted for binding against APRIL, BAFF or anti-tag antibody (3C8:2F4 Genentech, Inc.), and sequences of phage clones were analyzed as described previously [15]. One of the differences between the NMR ensemble of TACI_d2 and the crystal structure of the same domain bound to APRIL is the conformation of the C-terminal disulfide (Cys93/Cys104). A separate set of structures was calculated using dihedral angle restraints to force the disulfide bonds to adopt the conformation observed in the crystal structure (Ϫ60 Ϯ 20° for ␹1, Ϫ60° Ϯ 20° for ␹2, and Ϫ90 Ϯ 20° for ␹3) in addition to the NMR-derived restraints. Refinement of both structures was performed with the program REFMAC5 [40] and included TLS refinement and NCS restraints imposed separately on APRIL and the receptor, resulting in an R/Rfree of 16.7/20.3% for the APRIL1⁄7TACI_d2 structure and an R/Rfree of 17.8/21.3% for the APRIL1⁄7BCMA structure (Table S4)

RESULTS

DISCUSSION