Revealing Mechanism of Allostery in RIPK2 kinase

Receptor-interacting protein kinase 2 (RIPK2) regulates key signaling pathways in immune responses and inflammation, making it an intriguing target for treating inflammatory diseases such as inflammatory bowel disease. The interaction between RIPK2 and the E3 ligase X-linked inhibitor of apoptosis protein (XIAP) is pivotal, as resulting RIPK2 ubiquitination triggers the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, which are crucial for mounting an effective immune response. Previous research has established that the conformational dynamics of the RIPK2 modulates transition between active and inactive states. In the active state, RIPK2 binds to XIAP, whereas in the inactive state, this interaction is blocked. Building on these findings, our study introduces an innovative adaptive sampling protocol that integrates slow feature analysis with molecular dynamics simulations. This approach captures how the binding of RIPK2 kinase inhibitors allosterically modulates the conformational dynamics of RIPK2, leading to either stabilization of its active or inactive state. Predictions from our simulations, combined with results from a novel cell-based assay, demonstrate how a subset of RIPK2 kinase inhibitors modulate RIPK2-XIAP binding. Particularly, we show that targeting the hydrophobic back pocket of RIPK2's active site is crucial for modulating the active to inactive transition, resulting in inhibition of RIPK2-XIAP interactions. This study provides foundational insights into how small molecule binding regulates kinase-mediated protein-protein interactions and underscores the potential of using structural insights to design more precise and effective inhibitors.