Stoichiometry of LTbetaR binding to LIGHT.
LTbetaR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTalpha1beta2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTbetaRIgG, and monomeric LTbetaR protein binding to both LIGHT and LTalpha1beta2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTalpha1beta2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTbetaRIgG and LTbetaR 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 LTbetaRIgG bound to a LIGHT trimer, or two LTbetaR bound per LIGHT. Sedimentation velocity of varied ratios of LTbetaR 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 LTbetaR or LTbetaRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTbetaR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFbeta, 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 LTbetaR/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 LTbetaR rather than trimerization as expected from the structural paradigm.
Eldredge
John
J
Biogen Idec, Inc., 12 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Berkowitz
Steven
S
Corin
Alan F
AF
Day
Eric S
ES
Hayes
David
D
Meier
Werner
W
Strauch
Kathy
K
Zafari
Mohammad
M
Tadi
Madhavi
M
Farrington
Graham K
GK
D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002468:Cell Physiological Phenomena; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000378:metabolism; D008024:Ligands; D008233:Lymphotoxin-alpha / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D007074:Immunoglobulin G; D008024:Ligands; D008233:Lymphotoxin-alpha; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins