Assembly and structural properties of glucocorticoid-induced TNF receptor ligand: Implications for function

Abstract

Glucocorticoid-induced TNF receptor ligand (GITRL), a recently identified member of the TNF family, 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. Structural analysis reveals that the human GITRL (hGITRL) ectodomain self-assembles into an atypical expanded homotrimer with sparse monomer-monomer interfaces. Consistent with the small intersubunit interfaces, hGITRL exhibits a relatively weak tendency to trimerize in solution and displays a monomer-trimer equilibrium not reported for other TNF family members. This unique assembly behavior has direct implications for hGITRL-GITR signaling, because enforced trimerization of soluble hGITRL ectodomain results in an approximately 100-fold increase in its receptor binding affinity and also in enhanced costimulatory activity. The apparent reduction in affinity that is the consequence of this dynamic equilibrium may represent a mechanism to realize the biologically optimal level of signaling through the hGITRL-GITR pathway, as opposed to the maximal achievable level.

Fig. 1.

Structure of the hGITRL ectodomain shows an atypical expanded architecture not observed in conventional TNF family members. (A) Ribbon diagram of hGITRL monomer showing the classic jelly-roll fold of the THD. The β-strands are labeled, and the N and C termini are marked. (B) Superposition of hGITRL (red) and hTNF-α (blue; 1TNF-A) monomers shows significant differences in the strand and loop lengths. hGITRL trimer (C) and hTNF-α (1TNF) trimer (D) are shown in side view as ribbon diagrams. C and D clearly demonstrate that the hGITRL trimer possesses an atypical expanded assembly and lacks the typical compact architecture of the conventional THDs.

Fig. 2.

Receptor binding behavior of hGITRL. (A) Sensograms of the binding of hGITRL, at a range of concentrations (10,000 nM and 1.5-fold dilutions thereof) to immobilized hGITR. (B) The binding isotherm for the hGITRL–hGITR interaction was determined by fitting the steady-state binding data against a binding expression derived for the coupled reactions (i) monomer ↔ trimer for hGITRL self-assembly and (ii) trimeric hGITRL + hGITR ↔ trimeric hGITRL–hGITR complex. Curve fitting of the equilibrium binding data yields a K_d value of 60 ± 17 nM between the trimeric hGITRL and its receptor.

Fig. 3.

Enforced trimerization of hGITRL. (A) Molecular surface representation of the CC-hGITRL crystal structure shows that the fusion of the coiled-coil peptide to the N terminus of hGITRL does not induce any significant alterations in the global/local conformation of the protein. (B) Enforced trimerization of hGITRL augmented the in vitro T cell proliferative response. CD4⁺ T cells were stimulated with hGITRL variants (5 μg/ml) in the presence of 30 ng/ml anti-CD3. Data shown are the mean (± SEM) of quadruplicate cultures and are representative of at least three independent assays.

Fig. 4.

Receptor recognition surface of hGITRL. (A) Receptor binding activities of hGITRL mutants (125 nM) to immobilized hGITR. (B) hGITRL residues whose mutation affected receptor binding are mapped onto the hGITRL trimer and are colored accordingly. Orange and blue represent >90% and ≈50% reduction in binding, respectively. Alanine substitution of N106 (red) resulted in a ≈2- to 3-fold increase in receptor binding. (C) Surface representation of hGITRL-trimer, docked with a model of the hGITR monomer (shown in magenta). Areas on the surface of hGITRL that contribute to receptor binding are colored as described in B. (D) Alterations of the hGITRL–GITR interaction modulated T cell costimulation. The N106A mutation augmented the in vitro T cell proliferative response relative to the wild-type protein. The L65A-P66A-K68A triple mutant induced T cell proliferation similar to the PBS control. Data shown are the mean (± SEM) of quadruplicate cultures and are representative of at least three independent assays.