Metadynamics Simulation Reveals Allosteric Communication Effects of the Flipping Process of the Atypical DLG Motif in...

Kinases are key molecules in cell signal transduction. Their abnormal activation is closely related to cancer, inflammation, and metabolic diseases, making them important drug targets. However, the high sequence conservation of the kinase family limits the selectivity of inhibitors, and the dynamic conformational changes of kinases profoundly affect drug binding. Receptor-interacting protein kinase 1 (RIPK1) is a core factor in regulating cell necrosis. Its unique DLG (Asp-Leu-Gly) motif replaces the DFG (Asp-Phe-Gly) motif in traditional kinases. It is worth noting that, in the known RIPK1 crystal structure, the DLG motif is always in the "out" conformation, while its flipping mechanism and its regulatory mechanism on drug activity have not yet been elucidated. This study combined conventional molecular dynamics simulation and metadynamics simulation to deeply explore the conformational flipping process of the DLG motif in RIPK1 and its effect on protein conformation and drug binding. The results show that the flipping of the DLG motif occurs in coordination with the rotation of the αC helix, significantly changing the hydrophobicity and spatial volume of the ATP binding pocket, thereby regulating the affinity of drug molecules. In addition, the DLG flipping also reshapes the allosteric communication network of RIPK1, especially affecting the allosteric connection of the hinge region. The study further revealed the differential effects of different types of inhibitors on the conformational flipping of the DLG motif. This work not only provides a new structural perspective and theoretical basis for the design of highly selective RIPK1 inhibitors, but also provides important insights for the development of inhibitors targeting other kinases containing atypical DLG motifs.