Crystal structure of the m4-1BB/4-1BBL complex reveals an unusual dimeric ligand that undergoes structural changes upon 4-1BB receptor binding

Abstract

The interaction between the receptor 4-1BB and its ligand 4-1BBL provides co-stimulatory signals for T-cell activation and proliferation. However, differences in the mouse and human molecules might result in differential engagement of this pathway. Here, we report the crystal structure of mouse 4-1BBL and of the mouse 4-1BB/4-1BBL complex, which together provided insights into the molecular mechanism by which m4-1BBL and its cognate receptor recognize each other. Unlike all human or mouse tumor necrosis factor ligands that form noncovalent and mostly trimeric assemblies, the m4-1BBL structure formed a disulfide-linked dimeric assembly. The structure disclosed that certain differences in the amino acid composition along the intramolecular interface, together with two specific residues (Cys-246 and Ser-256) present exclusively in m4-1BBL, are responsible for this unique dimerization. Unexpectedly, upon m4-1BB binding, m4-1BBL undergoes structural changes within each protomer; moreover, the individual m4-1BBL protomers rotate relative to each other, yielding a dimerization interface with more inter-subunit interactions. We also observed that in the m4-1BB/4-1BBL complex, each receptor monomer binds exclusively to a single ligand subunit with contributions of cysteine-rich domain 1 (CRD1), CRD2, and CRD3. Furthermore, structure-guided mutagenesis of the binding interface revealed that novel binding interactions with the GH loop, rather than the DE loop, are energetically critical and define the m4-1BB receptor selectivity for m4-1BBL. A comparison with the human 4-1BB/4-1BBL complex highlighted several differences between the ligand- and receptor-binding interfaces, providing an explanation for the absence of inter-species cross-reactivity between human and mouse 4-1BB and 4-1BBL molecules.

Keywords: 4-1BB; 4-1BB ligand; CD137; CD137L; TNFRSF9; X-ray crystallography; cell surface receptor; immune cell; protein structure; protein–protein interaction; tumor necrosis factor (TNF).

Figure 1.

Architecture of m4-1BBL. A, domain organization of m4-1BB ligand. The construct used for crystallization is highlighted by dashed lines. Two N-linked glycosylation sites at positions 161 and 293 are highlighted. B, cartoon representation of m4-1BBL monomeric structure. The β-strands are labeled in accordance to the structure of h4-1BBL (PDB code 6D3N) and the N/C-terminal ends are marked. Asparagine residues of the potential N-glycosylation sites and N-glycans at Asn-161 position are shown as sticks. C, structural superposition of monomeric m4-1BBL (green) and h4-1BBL (orange) illustrates differences along the β-strands and the surface loops that connect these strands.

Figure 2.

Structure of m4-1BBL dimer. A, cartoon representation of m4-1BBL dimer with transparent molecular surface. The N- and C-terminal ends of both protomers are marked. The N-glycans at Asn-161 position are shown as ball and sticks. B, dimerization interface of m4-1BBL dimer. All residues mediating hydrophobic interactions at the lower interior of the dimer are shown as sticks in the inset below. A and B, carbon atoms of residues of protomer A are shown in yellow and protomer B are in green. In both panels, the cysteine residues (Cys-246) that form disulfide linkage between two protomers at the top of the dimer are shown as sticks. C, trimerization interface of h4-1BBL. The three protomers of h4-1BBL trimer are colored yellow, green, and cyan. The residues mediating salt bridge interactions and vdW contacts among all three protomers of h4-1BBL are labeled. D, structural alignment of residues mediating stabilization of monomer–monomer interface in h4-1BBL and m4-1BBL. The residues of m4-1BBL are shown in yellow with red labels and residues of h4-1BBL are shown in cyan with black labels. E, structure-based sequence alignment of F strand residues of conventional TNF ligands. In each ligand, the residue corresponding to Phe-199 of h4-1BBL is colored in red.

Figure 3.

Characterization of m4-1BBL dimerization mutants. A, SDS-PAGE (4–20%) analysis of purified WT and various mutants of m4-1BBL under nonreducing (−β-ME (lanes 1, 3, 5, and 7) and reducing conditions (+β-ME (lanes 2, 4, 6, and 8). Lanes 1 and 2 correspond to m4-1BBL (WT); lanes 3 and 4 correspond to m4-1BBL (S256F); lanes 5 and 6 correspond to m4-1BBL (C246S); lanes 7 and 8 correspond to m4-1BBL (C246S/S256F); lanes 9 and 10 represent deglycosylated (+PNGase) WT and C246S mutant of m4-1BBL. B and C, SEC-MALS analysis of WT and the S256F mutant of m4-1BBL. The molecular masses calculated from MALS data are indicated.

Figure 4.

Structure of m4-1BB/4-1BBL complex. A, crystal structure of tetrameric m4-1BB/4-1BBL complex. Two protomers of m4-1BBL are shown as transparent surface, and the m4-1BB receptors are shown as cartoon. The N- and C-terminal ends of receptor molecules are marked. B, binding interface between m4-1BBL and m4-1BB. The ligand is shown as green transparent surface. The four CRDs of m4-1BB are colored individually and labeled. C, detailed view of interactions between m4-1BBL and m4-1BB. The residues of m4-1BBL involved in the binding interface are shown as sticks in green color, and their respective loops are labeled. The residues of m4-1BB are colored according to their corresponding CRDs; CRD1 as cyan, CRD2 as light brown, and CRD3 as orange. The different types of modules in CRD2 and CRD3 are labeled. The hydrogen-bonding interactions are shown as black dashed lines, and the hydrophobic contacts are shown as magenta dashed lines.

Figure 5.

Receptor-induced conformational changes of m4-1BBL. A, superposition of free m4-1BBL monomer (yellow cartoon) with receptor-bound m4-1BBL (green cartoon) showing select conformational changes in the loops (blue), which are in contact with receptor. The m4-1BB is shown in light pink with its N- and C-terminal ends marked. Structural ordering in the DE and EF loops of m4-1BBL is highlighted in red. B, superposition of receptor-bound m4-1BBL dimer with free m4-1BBL dimer discloses the proper alignment of protomer A and the deviation in the orientation of protomer B. Protomers A and B of receptor-bound m4-1BBL are shown in green and orange and for free m4-1BBL are in yellow and blue. The EF loop that attains proper α-helix in both protomers induced by structural reordering of adjacent CD loop (blue) is highlighted in red. C, binding footprint of protomer B on protomer A revealing the dimerization interface of free m4-1BBL versus receptor-bound m4-1BBL. Residues participating in the dimeric interface of free m4-1BBL are colored blue and for receptor bound m4-1BBL are colored orange.

Figure 6.

SPR analysis for the binding of various mutants of m4-1BB with m4-1BBL. Single cycle kinetics to measure the interaction of m4-1BBL with immobilized m4-1BB-Fc variants on anti-Fc capture chip. SPR sensorgrams (red curve) were fitted by 1:1 binding model (black fit) to measure the binding affinity between m4-1BB variants and m4-1BBL. Experiments were performed in duplicate, and the on-rate (k_a), off-rate (k_d), and binding affinity (K_D) values with standard deviation were tabulated.

Figure 7.

Comparison of 4-1BB/4-1BBL complex in human and mouse. A, sequence alignment of THD regions of 4-1BBL of mouse and human. β-Strands present in m4-1BBL are labeled. The residues involved in binding interface of the complex are colored red, and the residues of the GH loop that define species selectivity for 4-1BBL are boxed. B, structural superposition of 4-1BB/4-1BBL complex in human and mouse by aligning the structurally equivalent β-strands of the ligand. 4-1BBLs of human and mouse are shown as a cartoon with transparent surface in cyan and green, respectively. 4-1BBs of human and mouse are represented as a cartoon in light pink and light blue. The four CRD regions of 4-1BB are labeled. C, interactions between GH loop residues and CRD2 region of m4-1BBL (green) and m4-1BB (blue, top left) and between h4-1BBL (cyan) and h4-1BB (pink, bottom left). The residues of h4-1BB that cause steric clash with Tyr-291 of m4-1BBL are shown as pink sticks, and the residues of m4-1BB that could bind to h4-1BBL are represented as blue sticks. D, structural deviation between CRD1 region of m4-1BB (light blue) and h4-1BB (light pink). The Arg-38 of m4-1BB CRD1 that makes salt bridge contact with Asp-170 of m4-1BBL (green) are shown as sticks. E, interactions between Asn-82 of m4-1BB and Tyr-291 of m4-1BBL; Phe-91 of m4-1BB with Phe-210 of m4-1BBL. The Asn-83 and Phe-92 of h4-1BB (pink sticks) have no interacting partners in h4-1BBL (cyan sticks). All the interactions are shown as black dashed lines.