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conserved substrate-binding cleft as observed in the crystal structure of cAPK:PKI complex (Figure
la,b). This finding allows the charges within this cleft to be predicted using the amino acid sequence of
any given kinase.
In the analysis of the structural data of other protein kinases, it is noted that only cAPK has been
crystallized with its specific peptide inhibitor. Nevertheless, three other structures of protein kinases
compared with the structure of the cAPK-PKI complex provide substantial evidence for the conservation
of the substrate binding cleft. The substrate binding cleft of the phosphorylase kinase structure has been
analyzed in detail and it is clear that all amino acids of the known specific substrate can be built into the
PKI model and all required corresponding charges can be found in the cleft of the phosphorylase kinase
structure. In the CK-1 structure determined without a peptide, the requirement of the peptide specificity
resides on the P-3 site, which has to be phosphorylated. An analysis of the surface charges of the cleft of
the CK-1 structure reveals the exact correspondence of the residues required to interact with a
phosphorylated substrate at this site.
Finally, the tripeptide of the pseudosubstrate site of IRK consisting of the Asp-Tyr-Tyr motif has
corresponding charges in the structure of the enzyme's substrate cleft and confirms the data obtained
from the degenerated peptide library for the unique sequence motifs of nine tyrosine kinases [22]. It is
becoming increasingly clear that the wealth of structural data of protein kinases with cAPK as a
prototype provides evidence for two important features concerning substrate binding. First, the substrate
binding cleft is structurally conserved and second, the surface charges of this cleft and hydrophobic
cavities on the surface are very diverse and correspond to the specificity requirement of the substrate for
individual protein kinases (see Figure la). It is now possible to use the structural conservation of the
substrate binding cleft to predict the charges and hydrophobic residues of the cleft to define substrate
specificities for individual kinases.
IV. Crystallographic Analysis of the ATP Binding Site Reveals Distinct Differences Utilized for the
Further Design of Specific Inhibitors
The diagram elucidating detailed interactions of ATP with the enzyme is presented in Figure 3a,b. Six
out of nine invariant residues of the catalytic core of protein kinase are involved in ATP binding and
catalysis. The key residues that hold the β and γ phosphates in position are the phosphate anchor, the
metal sites, and Lys168. The amides of the residues—Phe54, Gly55, and Ser53—are essential for the
position of the γ and β phosphates. The metal site coordinated by invariant Asp184 is also sequestered
by the β and γ phosphates and the metal
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