TβR-II Discriminates the High- and Low-Affinity TGF-β Isoforms via Two Hydrogen-Bonded Ion Pairs

The TGF-beta isoforms, TGF-beta 1, -beta 2,.and -beta 3, share greater than 70% sequence identity and are almost structurally identical. TGF-beta 2 differs from the others, however, in that it binds the TGF-beta type II receptor (T beta R-II) with much lower affinity than either TGF-beta 1 or -beta 3. It has been previously shown that three conserved interfacial residues, Arg25, Val92, Arg94, in TGF-beta 1. and -beta 3 are responsible for their high-affinity interaction with T beta R-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-beta 3 loss-of-function and TGF-beta 2 gain-of-function variants. One-dimensional H-1 NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for T beta R-II binding were determined by surface plasmon resonance biosensor analysis. Double substitutions revealed that nearly all of the high-affinity binding is contributed by Arg25 and Arg94. Single site substitutions showed that Arg94 makes the greatest contribution. Substitution of Arg25 and Arg94 with alanine verified the requirement of the arginine guanidinium functional groups for the highly specific hydrogen-bonded ion pairs formed between Arg25 and Arg94 of TGF-beta 1 and -beta 3, and Glu119 and Asp32 of T beta R-II. Further kinetic and thermodynamic analyses confirmed that Arg25 and Arg94 are primarily responsible for high-affinity binding and also revealed that noninterfacial longer range effects emanating from the TGF-beta structural framework contribute slightly to T beta R-II binding. Growth inhibition assays showed that binding changes generally correlate directly with changes in function; however, a role Val92 in this cellular context was uncovered.