title: TbetaR-II discriminates the high- and low-affinity TGF-beta isoforms via two hydrogen-bonded ion pairs.
abstract: The TGF-beta isoforms, TGF-beta1, -beta2, and -beta3, share greater than 70% sequence identity and are almost structurally identical. TGF-beta2 differs from the others, however, in that it binds the TGF-beta type II receptor (TbetaR-II) with much lower affinity than either TGF-beta1 or -beta3. It has been previously shown that three conserved interfacial residues, Arg25, Val92, Arg94, in TGF-beta1 and -beta3 are responsible for their high-affinity interaction with TbetaR-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-beta3 loss-of-function and TGF-beta2 gain-of-function variants. One-dimensional 1H NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for TbetaR-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-beta1 and -beta3, and Glu119 and Asp32 of TbetaR-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 TbetaR-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.
authors.affiliation: Biotechnology Research Institute, National Research Council, Montreal, Quebec H4P2R2, Canada.
authors.forename: Jason
authors.initials: J
authors.lastname: Baardsnes
authors.forename: Cynthia S
authors.initials: CS
authors.lastname: Hinck
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Hinck
authors.forename: Maureen D
authors.initials: MD
authors.lastname: O'Connor-McCourt
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000737:chemistry / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D053782:Transforming Growth Factor beta3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020033:Protein Isoforms; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D053782:Transforming Growth Factor beta3; D001120:Arginine; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II