Despite the efforts of pharmaceutical companies to develop specific kinase modulators, few drugs targeting kinases have been completely successful in the clinic. This is primarily due to the conserved nature of kinases, especially in the catalytic domains. Consequently, many currently available inhibitors lack sufficient selectivity for effective clinical application. Kinases phosphorylate their substrates to modulate their activity. One of the important steps in the catalytic reaction of protein phosphorylation is the correct positioning of the target residue within the catalytic site. This positioning is mediated by several regions in the substrate binding site, which is typically a shallow crevice that has critical subpockets that anchor and orient the substrate. The structural characterization of this protein-protein interaction can aid in the elucidation of the roles of distinct kinases in different cellular processes, the identification of substrates, and the development of specific inhibitors. Because the region of the substrate that is recognized by the kinase can be part of a linear consensus motif or a nonlinear motif, advances in technology beyond simple linear sequence scanning for consensus motifs were needed. Cost-effective bioinformatics tools are already frequently used to predict kinase-substrate interactions for linear consensus motifs, and new tools based on the structural data of these interactions improve the accuracy of these predictions and enable the identification of phosphorylation sites within nonlinear motifs. In this Review, we revisit kinase-substrate interactions and discuss the various approaches that can be used to identify them and analyze their binding structures for targeted drug development. The Catalytic Domain of Eukaryotic Protein KinasesTypically, eukaryotic protein kinases are composed of nonconserved regulatory domains and a conserved catalytic core of~250 amino acid residues that binds and anchors substrates and is responsible for catalysis (1). The catalytic domain consists of two lobes called N and C (also known as small and large lobes, respectively), named for their N-or C-terminal position, respectively, within the domain. The N-lobe consists of five-stranded, anti-parallel b sheets that are an essential part of the adenosine triphosphate (ATP) binding site, whereas the C-lobe is mostly helical (Fig. 1A). The active-site cleft, which contains the ATP binding site, lies between the two lobes (2). In an activated kinase, the lobes converge to form a deep cleft where the adenine ring of ATP binds such that the g-phosphate is positioned at the outer edge where the transfer of the phosphoryl group occurs, whereas the adenosine moiety is buried in a hydrophobic region of the pocket (Fig. 1B). Adjacent to the ATP binding pocket is a shallow crevice called the substrate binding site (SBS) that anchors the substrate and correctly positions the phosphorylatable residue (2).Catalysis is mediated by opening and closing of this active-site cleft. Substrates are anchored and p...

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