Stoichiometry of LTβR binding to LIGHT

LTβR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTα1β2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTβRIgG, and monomeric LTβR protein binding to both LIGHT and LTα1β2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTα1β2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTβRIgG and LTβR 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 LTβRIgG bound to a LIGHT trimer, or two LTβR bound per LIGHT. Sedimentation velocity of varied ratios of LTβR 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. Iso...