Structural basis and prediction of substrate specificity in protein serine/threonine kinases

Abstract

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serinethreonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.

Figure 1

Substrate-binding site in the crystal structure of PKA (9). The main chain of the protein is shown in worm representation, and the determinant residues (magenta), substrate peptide [green, individual substrate residues are labeled (−3) to (+3)], and ADP (blue) are shown in stick representation. The protein kinase surface is shown in transparent representation, determinant residues are marked 1–20, and individual protein kinase subsites are marked with yellow circles. The figure was produced with GRASP (43).

Figure 2

Schematic diagram of signaling connections linked to cell cycle control in S. cerevisiae. Yellow boxes, protein kinases; solid and dashed green arrowed connections, known and predicted activatory phosphorylations, respectively; solid and dashed red blocked connections, known and predicted inhibitory phosphorylations, respectively; circles, predicted sites in known substrates; open black arrows, general connections between processes. The joined boxes represent complexes. For the protein kinases analyzed (bold and underlined), all known interactions shown were also successfully predicted with PREDIKIN.

Figure 3

Schematic diagram of signaling connections linked to DNA damage checkpoints in S. cerevisiae, drawn as in Fig. 2. For the protein kinases analyzed (bold and underlined), all known interactions shown were also successfully predicted with PREDIKIN.