Conservation and divergence in the allosteric architectures of five human protein kinases

Protein kinases are crucial regulators in biology and are targets of over 100 approved drugs, but the conservation of their active sites makes specific inhibition difficult. Targeting allosteric secondary sites is a promising strategy to improve selectivity, reduce toxicity, and overcome resistance mutations, yet these sites are largely unknown for most kinases. This study addresses that gap by quantifying the activity and abundance of more than 160,000 variants of five human kinase domains—SRC, FGR, JNK2/MAPK9, ZAK/MAP3K20, and TSSK2—to construct complete energetic and allosteric maps. The results show that while all five kinases have distance-dependent but anisotropic allostery, each has a distinct allosteric architecture, surface, and unique set of pockets for therapeutic targeting. Some functional secondary sites are conserved across all five, but others are protein-specific or even switch between inhibitory and activating roles depending on the kinase. This divergence is especially pronounced in activation-linked allosteric networks. The findings imply that allosteric regulation is highly individualized across kinases, meaning that effective allosteric drugs must be tailored to each kinase's unique landscape rather than relying on generic targets. This resource could accelerate development of next-generation kinase inhibitors and activators with improved specificity and fewer off-target effects.