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

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DOIResolve DOI: http://doi.org/10.1021/bi8019004
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Journal titleBiochemistry
Pages21462155; # of pages: 10
AbstractThe TGF-isoforms,TGF-1,-2,and-3,share greater than 70% sequence identity and are almost structurally identical. TGF-2 differs from the others, however, in that it binds the TGF-type II receptor(TR-II)with much lower affinity than either TGF-1or-3. It has been previously shown that three conserved interfacial residues,Arg25,Val92,Arg94,in TGF-1 and-3 are responsible for their high-affinity interaction with TR-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-3 loss-of-function and TGF-2 gain-of-function variants. One-dimensional 1H NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for TR-II binding were determined by surface plasmon resonance biosensor analysis. Double substitutions revealed that nearly all of the highaffinity 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-1 and-3, and Glu119 and Asp32 of TR-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-structural framework contribute slightly to TR-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.
Publication date
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute
Peer reviewedYes
NRC number50691
NPARC number16526902
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Record identifierf930d7a2-1fcb-4c57-a6b0-b6dad3c59cc6
Record created2010-12-14
Record modified2016-05-09
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