Crystal structure of the human 4-1BB/4-1BBL complex

Abstract

4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-Å resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.

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

“…Unique dimeric organization of m4-1BB ligand-To date, all the characterized conventional human or mouse TNF ligands organize into a symmetrical trimeric bell shape to form a functional biological unit. Recently, we and others reported the structure of h4-1BBL that also assembles as a trimeric bell shape despite its low sequence similarity with conventional members (11,20). However, m4-1BBL self-assembled as a two-fold symmetrical homodimer, in which both protomers are covalently connected by a disulfide bond ( Figure 2A).…”

“…In the complex, a single m4-1BBL engages a monomeric receptor leading to the formation of a tetrameric ligand-receptor complex. In all other reported TNF-TNFR complexes, including the recently solved structure of the human 4-1BBL/4-1BB complex (11,20), each receptor binds between two adjacent ligand protomers. M4-1BB, however, exclusively binds to one ligand protomer (protomer A) with no obvious contact with the adjacent subunit (protomer B) ( Figure 4A).…”

“…Based on sequence diversity, human 4-1BBL was previously categorized within the latter group. However, two recent crystal structures of h4-1BBL revealed that h4-1BBL forms a compact bell shaped trimer characteristic of conventional TNF ligands (11,20). All conventional ligands bind to their cognate receptors in a similar manner and they all contains a conserved hydrophobic residue that acts as a 'hot spot' in their DE loop that is shown to be energetically important for receptor binding (13).…”

“…Interestingly, although human 4-1BBL exhibits all the features of the conventional ligands, the hydrophobic residues in the DE loop are not conserved. Additionally, recent crystal structures showed that the DE loop residues are not contributing towards binding affinity, suggesting that h4-1BB although forming a bellshaped trimer is still unique in its interaction with h4-1BBL (11,20,21).…”

“…While many TNF-TNFR complexes had been crystallized, until recently, very little was known about 4-1BB/4-1BBL interactions. Our recent characterization of the interaction between human 4-1BB and 4-1BBL led us to hypothesize a distinct mechanism of h4-1BB signaling, in which covalent receptor-dimerization would favor the formation of a 2D-signaling network to initiate robust signaling (11,20). Adding to this, our earlier biochemical studies suggested that recombinant m4-1BBL could form a covalent dimer rather than the conventional trimer (12).…”