Impact of R361H SMAD4 Mutation on TGF-ß Signaling in Colorectal Cancer

Introduction: SMAD4 mutations disrupt TGF-ß signaling in colorectal cancer. We focus on the recurrent MH2-domain R361H mutation, whose structural and interaction effects remain unclear. Using structural modeling, docking, and molecular dynamics, we assess how R361H alters SMAD4 stability and binding to SMAD1/2/3/5. Methodology: AlphaFold models of SMAD1-5 were obtained from RCSB, and the R361H mutation was introduced to SMAD4 with PyMOL. Wildtype and mutant SMAD4 were docked separately to each R-SMAD with ClusPro. The resulting complexes were subsequently repaired, solvated, and parameterized in GROMACS. Molecular dynamics simulations were run for 100 ns per complex. Global and domain-specific RMSD/RMSF were computed with GROMACS. MMPBSA.py was used to calculate binding energies and per-residue contributions. Results: R361H mutation altered SMAD4-R-SMAD complex stability in a partner-specific way. At the complex level, SMAD4-SMAD1 and SMAD4-SMAD3 showed only small changes in overall RMSD (Δcomplex ≈ −0.05 nm for SMAD1 and +0.07 nm for SMAD3, MUT - WT). In contrast, SMAD4-SMAD2 and SMAD4-SMAD5 were clearly destabilized, with much larger RMSD shifts (Δcomplex ≈ +0.34 and +0.50 nm, respectively).Across all four complexes, R-SMAD linkers became more flexible in the mutant (+0.10-0.34 nm) while SMAD4 MH2 mobility increased in the SMAD1, SMAD3, and SMAD5 complexes.RMSF analysis localized these changes to SMAD4 residues ∼470-490 and 540-550 and to partner-specific regions in each R-SMAD.Energy decomposition revealed distinct mechanisms for each partner. In the SMAD1-SMAD4 complex, strongly interacting residues in wildtype and mutant showed minimal overlap; PyMOL alignment (RMSD ≈ 36 Å) and docking scores indicate that R361H drives an alternative, remodeled binding mode rather than simply weakening the wildtype interface. In the SMAD2-SMAD4 complex, a hydrophobic to polar patch interaction (SMAD4 274-281, SMAD2 181-187) becomes less favorable in the mutant, consistent with increased SMAD4 RMSF and higher complex RMSD. In the SMAD3-SMAD4 complex, a key R420SMAD3-E417SMAD4 salt bridge is weakened by ∼3 kcal/mol in the mutant. RMSF data indicate that in the wildtype, a flexible C-terminal tail frequently adopts the orientation needed for this interaction, whereas the mutant tail is more rigid in a less favorable orientation. In the SMAD5-SMAD4 complex, the mutant gains favorable contacts between SMAD5 residues 185-188 and a polar SMAD4 patch (263-268), with increased SMAD4 MH2 mobility (∼0.2 nm). Conclusion: This framework suggests that R361H remodels SMAD4-R-SMAD interfaces in a partner-specific manner, inducing marked reorganization with SMAD1 and more nuanced shifts in SMAD2/3/5. These partner-specific interfaces may represent druggable surfaces for restoring tumor-suppressive TGF-ß responses or selectively constraining pro-metastatic signaling in R361H-mutant colorectal cancer. Citation Format: Evan Boczko, Isaac Silverman, Yisrael Wiener, Sanjay Goel, Radhashree Maitra. Structural and kinetic effects of R361H SMAD4 mutation on TGF-ß signaling in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5441.