Structural Basis for TGF-β Mimetic Peptide-Induced Signaling Activation Through Molecular Dynamics Simulations.

Transforming growth factor-β (TGF-β) mimetic peptides offer significant therapeutic potential due to their superior pharmacological properties over the native cytokine. Our previous work identified two such peptides, TB1 and TB2, which bind to the type II TGF-β receptor (TβRII) yet elicit distinct cellular responses. To uncover the mechanistic basis for the functional divergence, we employed integrated molecular dynamics (MD) simulations with the AlphaFold3-predicted structures. Our analytical results indicated that TB2 stabilizes a dynamic complex with TβRII and is predicted to facilitate type I receptor (TβRI) engagement possibly involving a critical hydrogen bond between TB2-Gly11 and TβRI-Phe60. The resulting trimeric assembly (TB2-TβRII-TβRI) exhibits a higher relative binding affinity (-67.76 ± 7.70 kcal/mol) and structural stability. In contrast, the TB1-TβRII complex fails to productively engage TβRI. These computational results were experimentally validated. Western blot analysis confirmed that TB2, but not TB1, activates the canonical TGF-β/Smad pathway by enhancing the expression and phosphorylation of Smad3. This study will elucidate the dynamic structural basis for the activity of TGF-β mimetic peptides and suggest TB2 as a promising lead candidate for the rational design of tissue-regenerative therapeutics.