| Transforming Growth Factor beta (TGF-beta), a ∼25 kDa dimeric protein, is a multifunctional cytokine that regulates many aspects of cell activities. TGF-beta also inhibits cancer progression in some cases but stimulates tumor growth in others. We investigated the receptor-binding and biological activities of synthetic peptides of TGF-beta, which mimic the binding motif of TGF-beta to its receptors. Some of these peptides were found to be agonists of TGF-beta in in vitro cellular proliferation assays. The peptide sequences were based on amino acid residues of TGF-beta that were previously identified as being critical for bioactivity. The TGF-beta peptides containing residues 87--100 of TGF-beta1, -beta2 and -beta3 were synthesized by solid phase peptide synthesis and purified to >95% purity using HPLC. A disulfide bond was engineered to provide global conformational constraints. ELISAs and flow cytometry experiments showed that the constrained peptides retain binding activities toward the ectodomain of the type III receptor, and some peptides even bound to the ectodomain of the type II receptor. Substitution of alanine for residues Tyr91, Lys94 and Lys97 of TGF-beta2 demonstrated the importance of these residue in receptor binding. We screened a phage display peptide library against the immobilized synthetic TGF-beta2 peptide. The data suggested the beta-hairpin motif in TGF-beta as a binding site of thrombospondin-1, which activates latent TGF-beta. We also resolved the aqueous structure of the cyclic TGF-beta3 peptide using 2D NMR and found a beta-hairpin conformation which was expected and is consistent with previous similar data of synthetic peptides of TGF-beta1 and beta2. In addition, we conducted molecular dynamics simulations on theoretical and NMR models of the TGF-beta peptides. The results revealed that the intrinsic stability of the beta-hairpin conformation is the structural basis of the binding and biological activities of TGF-beta peptides. |
