Enzymes catalyze the reactions of life and are the targets of many drugs. Most inhibitors bind conserved active sites, frequently lacking specificity. Targeting allosteric sites can increase specificity, reduce toxicity, and allow fine-tuning of activity; however, most allosteric sites in enzymes are unmapped. Here, we present a comprehensive experimental allosteric map of the Src protein kinase. We quantify the effects of more than 50,000 single and double amino acid substitutions on activity and abundance and use thermodynamic modeling to disentangle changes in fold stability and catalysis. The comprehensive energy landscape reveals that allostery across the kinase domain is extensive, directionally biased, and modulated by its regulatory domains. Inhibitory—but not activating—allosteric mutations show a strong distance-dependent decay away from the active site. Using the map, we identify multiple potentially druggable allosteric sites not previously reported in Src or other kinases. Our results establish a framework for comprehensive mapping of allostery in kinases and other enzymes important for medicine and biotechnology.
Beltran et al. (2026) studied this question.