Overall structure of the 4-1BBL monomer. The structure shows a two-layered jellyroll β-sandwich topology similar to the canonical structure of other TNF family members. The inner and outer sheets of the jellyroll β-sandwich are composed of A′HCF and ABGDE, respectively. The long A′B loop is colored in yellow.

4-1BBL forms a trimer that is atypical for members of the TNF family. a, top view of the 4-1BBL trimer shown in ribbon representation. 4-1BBL forms a 3-fold symmetric assembly resembling a three-bladed propeller. b, side view of the 4-1BBL trimer. c, crystal structure of GITRL in ribbon representation (Protein Data Bank code 2Q1M). The shape of the GITRL trimer resembles a blooming flower. d, crystal structure of TNFα (Protein Data Bank code 1TNF), which shows the canonical bell-shaped trimer. e, the interactions between 4-1BBL subunits in the trimer are primarily mediated by hydrophobic interactions between the C-terminal tail of one subunit and the A′B loop and the A′, C, and F strands of the adjacent subunit. The residues involved in the hydrophobic interactions are: Gln⁸⁹ (AA′ loop), Val¹⁴⁰ (C strand), Leu²⁰³ (F strand), His²⁰⁵ (F strand), Val²⁴⁰ (C-terminal tail), Thr²⁴¹ (C-terminal tail), Pro²⁴² (C-terminal tail), and Pro²⁴⁵ (C-terminal tail) in one protomer (blue) and Gln⁹⁴ (A′ strand), Phe⁹² (A′ strand), Leu¹¹⁵ (A′B loop), Tyr¹⁴² (C strand), Phe¹⁴⁴ (C strand), Phe¹⁹⁷ (F strand), and Phe¹⁹⁹ (F strand) in the adjacent protomer (green).

Comparison of the structures of 4-1BBL and TNFα. a and b, superimposition of 4-1BBL (light blue) and TNFα (wheat). The A′B loop in 4-1BBL is colored in light green, and the AA′ loop in TNFα is colored in orange. The 4-1BBL structure has a much longer A′B loop because of its lack of strands corresponding to the A′ and B′ strands in the TNFα structure. c, positions of the N and C termini in 4-1BBL and TNFα.

Binding affinity of the 4-1BBL-4-1BB interaction measured by FACScan. a, control experiments using phycoerythrin-conjugated anti-4-1BBL monoclonal antibody as positive control and isotype-control antibody as negative control. b, measurements of binding affinities of wild-type and mutant 4-1BBL for 4-1BB. Mutations L115G, K127A, and Q227A decreased the binding affinity, whereas mutations R171G and S172G showed no effect. The Q89A mutation resulted in a 40% decrease in binding affinity.

Model of the 4-1BBL-4-1BB complex showing the interaction sites of 4-1BBL and 4-1BB. The structure of the 4-1BBL-4-1BB complex was modeled based on the known structure of the TNFβ-TNFR1 complex (Protein Data Bank code 1TNR). 4-1BBL residues involved in receptor binding, Leu¹¹⁵, Lys¹²⁷, and Gln²²⁷, are labeled and colored in orange.

Electron microscopy of 4-1BBL and its complex with 4-1BB. a, raw image of negatively stained 4-1BBL and representative class averages (panels 1–5), which indicate a trimeric organization of 4-1BBL in solution. b, raw image of the negatively stained 4-1BBL-4-1BB complex and representative class averages of these complexes (panels 1–5). The scale bar represents 25 nm, and the side length of the individual panels is 18 nm. c, the 4-1BBL trimer in the orientation used to calculate the projection shown in panel 6 of A. d, model of the 4-1BBL-4-1BB complex in the orientation used to calculate the projection shown in panel 6 of B. The 4-1BBL subunits are colored in blue, pink, and green, and the 4-1BB subunits are yellow, red, and cyan. The 4-1BB structure was modeled based on the structure of DR5 (Protein Data Bank code 1DU3). The 4-1BBL-4-1BB complex has a windmill-like appearance, which is consistent with the EM averages of the complex.