Crystal structure of murine 4-1BB and its interaction with 4-1BBL support a role for galectin-9 in 4-1BB signaling

Abstract

4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat-type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)-deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution. We found that similar to other TNFRSFs, m4-1BB has four cysteine-rich domains (CRDs). However, the organization of CRD1 and the orientation of CRD3 and CRD4 with respect to CRD2 in the m4-1BB structure distinctly differed from those of other TNFRSFs. Moreover, we mapped two Asn residues within CRD4 that are N-linked glycosylated and mediate m4-1BB binding to Gal-9. Kinetics studies of m4-1BB disclosed a very tight nanomolar binding affinity to m4-1BBL with an unexpectedly strong avidity effect. Both N- and C-terminal domains of Gal-9 bound m4-1BB, but with lower affinity compared with m4-1BBL. Although the TNF homology domain (THD) of human (h)4-1BBL forms non-covalent trimers, we found that m4-1BBL formed a covalent dimer via 2 cysteines absent in h4-1BBL. As multimerization and clustering is a prerequisite for TNFR intracellular signaling, and as m4-1BBL can only recruit two m4-1BB monomers, we hypothesize that m4-1BBL and Gal-9 act together to aid aggregation of m4-1BB monomers to efficiently initiate m4-1BB signaling.

Keywords: N-linked glycosylation; X-ray crystallography; cell surface receptor; galectin; protein structure; protein-protein interaction; recombinant protein expression; site-directed mutagenesis; surface plasmon resonance (SPR); tumor necrosis factor (TNF).

Figure 1.

Expression of m4-1BB constructs. A, domain organization of m4-1BB. Signal P, signal peptide; pCRD1, partial CRD1; TM, transmembrane region; CR, cytoplasmic region containing TRAF binding motif. *, potential N-linked glycosylation sites indicated. B, size exclusion chromatogram of wildtype (WT) m4-1BB (blue line) and molecular weight marker as a reference (red line). C, Coomassie-stained 4–20% SDS-PAGE analysis of purified proteins of m4-1BB (WT), m4-1BB N128A mutant, and m4-1BB N138A mutant with (−PNGase) or without (+PNGase) N-linked glycans. Molecular weights of protein standards are indicated.

Figure 2.

Structure of m4-1BB. A, ribbon representation of m4-1BB with transparent molecular surface colored differently for each cysteine-rich domain. CRD1, blue; CRD2, magenta; CRD3, cyan; CRD4, green. All cysteine residues and Asn residues that undergo N-linked glycosylation are shown as sticks. B, superposition of all monomers of 4-1BB from the three different space groups P2₁, P2₁2₁2, and P4₃, showing the well ordered overall structure as well as the variation in the relative spatial orientation of N-glycans at Asn-138 position. C, representative 2F_o − F_c electron density map contoured at 1σ, for N-glycans at positions 128 and 138 within the CRD4 region. All N-linked glycans at Asn-128 and Asn-138 are shown as sticks. All figures were made in PyMOL.

Figure 3.

Organization of type A and B modules. A, close up view of CRD2 of m4-1BB showing A1 and B2 modules with their disulfide connectivity. B, schematic representation of A2 module present in CRD3 of m4-1BB. C, structural alignment of A2 subdomain of m4-1BB and TNFR2 (PDB code 3ALQ). m4-1BB is shown in blue and TNFR2 is gray. In all panels, the sulfur atoms of disulfide linkages are shown as yellow sticks. All figures were made in PyMOL.

Figure 4.

Superimposition of m4-1BB with other TNFRSF members. Structural alignment of m4-1BB (red color) with various TNFRSF members (green color) based on the Cα atoms of CRD2 region reveals structural differences at CRD1, CRD3, and CRD4 regions. PDB codes of crystal structures of TNFR members used for superposition are as follows: TNFR1, 1TNR; TNFR2, 3ALQ; LTbR, 4MXW; OX40, 2HEV; RANK, 3ME4; DcR3, 3MHD; TRAILR2, 4I9X; CD40, 5DMJ; HVEM, 4FHQ; DR6, 3QO4). All figures were made in PyMOL.

Figure 5.

Expression of m4-1BBL and binding interaction with m4-1BB. A, schematic representation of m4-1BBL depicting the CR (cytoplasmic region), transmembrane region (TM), and THD. *, potential N-linked glycosylation sites Asn-161 and Asn-293, as well as two extra cysteines (compared with h4-1BBL) in the THD region are indicated. B, SEC profile of purified m4-1BBL 140–309 (blue line) compared with a protein standard with molecular mass in kDa (red line). C, SDS-PAGE of m4-1BBL under non-reducing (−β-ME) and reducing (+β-ME) conditions. Lane 5 corresponds to m4-1BBL treated with PNGase F to remove N-glycans, which also reduces the protein. D and E, mass spectrometric analysis of m4-1BBL. D, ESI of wildtype m4-1BBL (140–309) determines the molecular mass of ∼43.7 kDa. E, ESI spectra for m4-1BBL subjected to standard reduction and S-carboxymethylation reaction confirmed the reduction of disulfide-linked dimer to a monomer with half the molecular mass (∼21.9 kDa).

Figure 6.

SPR binding analysis of m4-1BBL and Gal-9 to m4-1BB. Single cycle kinetic SPR sensorgram depicts the sequential binding response of increasing concentrations of monomeric m4-1BB (A) or dimeric m4-1BB-Fc (B) to assess avidity contribution in apparent binding affinity. C, single cycle kinetics to measure the interaction of dimeric m4-1BBL to immobilized m4-1BB-Fc. D and E, SPR sensorgrams for the interaction of m4-1BB (WT) with CTD (D) and NTD (E) of Gal-9. In all sensorgrams, the experimental data are shown as a red trace and the 1:1 fitted model as a black solid line.