Enzymes catalyze the reactions of life and are the targets of nearly all small molecule drugs. Most drugs inhibit enzymes by binding to conserved active sites, causing problems of specificity and toxicity. Targeting regulatory allosteric sites can increase specificity, overcome drug resistance and tune or activate activity.However, the vast majority of enzymes have no known allosteric sites and methods do not exist to globally identify or predict them.Here we present a general and fast method to globally chart allosteric communication in enzymes and apply it to the Src protein kinase to produce the first comprehensive map of negative and positive allosteric control of enzymatic activity. Allosteric control of Src is pervasive, anisotropic and distance dependent, but fairly predictable from simple sequence and structural features. The comprehensive allosteric map enables unbiased identification of all the allosterically active surface pockets of the Src kinase for the development of activatory and inhibitory drugs.This general approach can be used to chart global allosteric maps of many kinases, enzymes, and other biochemical activities important for medicine and biotechnology.
Overall design
We designed a variant library covering the kinase domain of the human tyrosine kinase Src. The library is split in 5 blocks of ~60 aa residues, and each block consists of all single substitutions in 10 genetic backgrounds (54,455 genotypes in total across the full kinase domain). Each block was cloned into a Src kinase domain alone construct (KD), and a full-length Src construct (FL), and each of these was selected in triplicate in activity and protein abundance assays in yeast. The data files here correspond to the amplicon-seq libraries of the population of variants in each block before and after selection (inputs and outputs).
Less...
More...