Minimal Perturbation of Activation Loop Dynamics Rewires Kinase Signaling

Enzymes are central to life, with their catalytic activity often shaped by the dynamic conformations of regulatory loops. In hub enzymes such as tyrosine kinases, the activation loop critically controls substrate specificity, catalytic efficiency, and downstream signaling, shaping cellular fate. Yet, the molecular mechanisms by which loop dynamics encode these functions remain incompletely understood. Here, we used SRC kinase as a model to dissect how minimal perturbations of the activation loop reprogram kinase behavior. By generating and characterizing multiple variants, we identified a triple-deletion mutant with altered loop dynamics. Structural and biochemical analyses revealed that this variant explores distinct loop conformations and exhibits a subtle shift in substrate preference toward more acidic motifs. These fine-tuned conformational changes translated into specific cellular signaling outcomes, as demonstrated by phosphoproteomic profiling. Comparative analysis across species further showed that nature exploits similar loop remodeling strategies to modulate kinase function. Together, our findings provide a blueprint for rationally tuning kinase activity and offer a generalizable framework for rewiring signaling pathways in diverse cellular contexts.