Molecular dynamics simulations for understanding dual ubiquitination mechanisms to consider S-phase kinase-associated protein 2 (SKP2) as a potential drug target in breast cancer.

Introduction: Dual ubiquitination mediated by S-phase kinase-associated protein 2 (SKP2) plays a critical role in breast cancer progression by directing substrates toward either proteasomal degradation or signaling activation. However, the structural determinants governing SKP2 recognition of different ubiquitin-linked substrates, particularly p27 and Akt1, remain poorly understood. Methods: To investigate this mechanism, integrative computational approaches including protein-protein docking, molecular dynamics simulations, hydrogen-bond analysis, radius of gyration (Rg), root mean square deviation (RMSD), and MM/PBSA free-energy calculations were employed. Isopeptide bonds were constructed between the C-terminal Gly76 of ubiquitin and Lys134 of p27 (K48-linked) or Lys135 of Akt1 (K63-linked) to represent the post-ubiquitin transfer stage and preserve native ubiquitin conjugation geometry. Results: Computational analyses revealed distinct substrate recognition patterns by SKP2. The p27 complex adopted a compact interaction interface favorable for selective recognition, whereas Akt1 displayed a broader interaction surface supporting stable engagement. Docking studies demonstrated strong SKP2 interactions with UbK48-p27 and UbK63-Akt1 complexes, yielding HADDOCK scores of -103.8 and -85.5, respectively. Molecular dynamics simulations showed that UbK48 induced structural loosening in the SKP2-p27 complex (RMSD 1.61 nm; Rg 3.57 nm), consistent with proteasomal targeting, while UbK63 maintained a compact and stable SKP2-Akt1 assembly (RMSD 0.86 nm; Rg 3.33 nm), supporting sustained signaling activity. Hydrogen-bond and MM/PBSA analyses further demonstrated destabilization of SKP2-p27 (10.96 H-bonds; -61.63 kcal/mol) and stabilization of SKP2-Akt1 (14.42 H-bonds; -88.69 kcal/mol). Discussion: This study provides the first atomistic evidence of isopeptide-mediated dual ubiquitination by SKP2. The findings identify distinct structural determinants that govern substrate-specific outcomes toward degradation or activation, revealing potential regulatory interfaces within SKP2-substrate complexes. These insights establish a framework for developing computationally prioritized strategies to design targeted inhibitors aimed at disrupting oncogenic signaling and overcoming SKP2-driven drug resistance in breast cancer.