Evolution of GITRL immune function: murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily

Abstract

Glucocorticoid-induced TNF receptor ligand (GITRL), a recently identified member of the TNF superfamily, binds to its receptor, GITR, on both effector and regulatory T cells and generates positive costimulatory signals implicated in a wide range of T cell functions. In contrast to all previously characterized homotrimeric TNF family members, the mouse GITRL crystal structure reveals a previously unrecognized dimeric assembly that is stabilized via a unique "domain-swapping" interaction. Consistent with its crystal structure, mouse GITRL exists as a stable dimer in solution. Structure-guided mutagenesis studies confirmed the determinants responsible for dimerization and support a previously unrecognized receptor-recognition surface for mouse GITRL that has not been observed for any other TNF family members. Taken together, the unique structural and biochemical behavior of mouse GITRL, along with the unusual domain organization of murine GITR, support a previously unrecognized mechanism for signaling within the TNF superfamily.

Fig. 1.

Structure of the mouse GITRL ectodomain shows a previously unrecognized dimeric assembly not observed in conventional TNF family members. (A) Amino acid sequence alignment of the mouse and human GITRL ectodomains. β-Strands identified in the mouse GITRL structure are indicated and labeled. Positions of the Cys residues that form the disulfide bond in both mouse and human GITRL are indicated with red arrows. Mouse GITRL residues mediating the domain swap and forming the potential hydrogen bonds at the monomer–monomer interface are marked with red asterisks and blue dashes, respectively, on the top of the alignment. Residues forming potential contacts at the intersubunit interfaces of human GITRL trimer are marked with black asterisks at the bottom of the alignment. (B) Ribbon diagram of the mouse GITRL monomer showing the classical jelly-roll fold of the TNF homology domains. The β-strands are labeled, and the N and C termini are marked. (C) Superposition of the mouse (red) and human (blue) GITRL shows significant differences in the arrangements of the A′B′ and CD loops and the C termini. (D) The mouse GITRL dimer is composed of two monomers, shown in red and green, that are associated with an angle of ≈40° with respect to each other. The C terminus of each subunit participates in a domain swap with the other subunit and is highlighted with a black box. (E) Detailed view of the mouse GITRL domain-swap interaction. Mouse GITRL residues, H64 and P68 from one monomer and P170, F171, and I172 from the other, are shown. P170, F171, and P68 are arranged to form a strong hydrophobic stacking interaction. The human GITRL residues at the corresponding positions (Q for H64, S for P68, and Q for P170) are indicated in the parentheses.

Fig. 2.

Mouse GITRL shows a stable dimeric assembly in solution. (A) Elution profile of soluble mouse GITRL (refolded from inclusion bodies in E. coli) from Superdex G-75 gel-filtration column. The elution positions of size calibration standards of 25 kDa and 43 kDa are indicated. (B) The values of S_20,w estimated for refolded mouse GITRL from sedimentation velocity analysis as a function of total monomer concentration at 5°C (●). The open circle and open triangle indicate the S values calculated from the crystal structure for dimer and monomer (top to bottom, respectively). ▴ and ▾ represent the values of S_20,w estimated for the refolded humanized mouse GITRL and the murinized human GITRL, respectively, as a function of total monomer concentration at 25°C.

Fig. 3.

Receptor binding behavior of mouse GITRL. (A) Sensograms of the binding of mouse GITR (purified from HEK293T cells) at a range of concentrations (4,860 nM and 2-fold dilutions thereof) to immobilized mouse GITRL (expressed in E. coli). (B) Nonlinear 1:1 Langmuir fitting of the steady-state binding data yields a K_d of 4.37 ± 0.15 μM.

Fig. 4.

Comparison of the receptor-recognition surfaces of mouse and human GITRL. Molecular surfaces of mouse (A) and human (B) GITRL show the arrangement of the surface exposed loops AA′, DE, and GH. Mutations of the loops AA′ (mutant S59A-S60-K62A; orange), DE (mutant K116A-N117A-D118A; red), and GH (mutant Q155A-K156A-T157A; blue) in mouse GITRL did not alter its receptor binding affinity, whereas corresponding mutations in the human ortholog significantly alter the receptor binding ability of the protein (15). Residues from these loops are closely clustered in the human GITRL, forming its receptor-recognition surface. In the mouse GITRL dimer, the corresponding residues are distantly located, suggesting a noncanonical receptor binding surface. Ribbon diagrams are shown on the right to indicate the orientation of the molecules.

Fig. 5.

Unique dimeric assembly and domain organization of mouse GITRL/GITR suggests a previously unrecognized mode of ligand:receptor interaction in the TNF/TNFR superfamily. (A) In conventional TNF:TNFR complexes, each receptor monomer (containing CRDs 1, 2, 3, and 4) binds at the cleft between the two adjacent subunits of the ligand homotrimer. TNFR CRDs 2 and 3, which typically contribute to ligand binding, are indicated with arrows. (B) Human GITR with CRDs 2, 3, and 4 is shown in complex with the human GITRL trimer. The human GITRL trimer, despite having an atypical expanded assembly, utilizes a canonical receptor binding surface similar to the conventional TNF ligands. (C) Dimeric mouse GITRL appears to possess a noncanonical receptor binding surface. This is consistent with the atypical domain organization of mouse GITR. Mouse GITR contains CRDs 1, 3, and 4 and lacks the signature of CRD 2, which critically contributes to the ligand binding surface(s) of the classical TNFRs.