Stoichiometry of LTβR binding to LIGHT

LT beta R is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LT alpha 1 beta 2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LT beta RIgG, and monomeric LT beta R protein binding to both LIGHT and LT alpha 1 beta 2 using surface plasmon resonance and found values of < 0.1 and 38 nM for LIGHT and < 0.1 and 48 nM for LT alpha 1 beta 2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LT beta RIgG and LT beta R binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC-LS corresponded to a single LT beta RIgG bound to a LIGHT trimer, or two LT beta R bound per LIGHT. Sedimentation velocity of varied ratios of LT beta R to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LT beta R or LT beta RIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LT beta R to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNF beta, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor-ligand complexation paradigm for the entire TNF family. The LT beta R/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LT beta R rather than trimerization as expected from the structural paradigm.