Crystal structure of a cAMP-dependent protein kinase mutant at 1.26 Å:: New insights into the catalytic mechanism

The catalytic subunit of cAMP-dependent protein kinase has served as a paradigm for the entire kinase family. In the course of studying the structure-function relationship of the P + 1 loop (Leu198-Leu205) of the kinase, we have solved the crystal structure of the Tyr204 to. Ala mutant in complexes with Mg.ATP and an inhibitory peptide at 1.26 Angstrom, with overall structure very similar to that of the wild-type protein. However, at the nucleotide binding site, ATP was found largely hydrolyzed, with the products ADP-PO4 retained in the structure. High-resolution refinement suggests that 26% of the molecules contain the intact ATP, whereas 74% have the hydrolyzed products. The observation of the substrate and product states in the same structure adds significant information to our understanding of the phosphoryl transfer process. Structural examination of the mutation site substantiates and extends the emerging concept that the hydrophobic core in the large lobe of the kinase might serve as a stable platform for anchoring key segments involved in catalysis. We propose that Tyr204 is critical for anchoring the P + 1 loop to the core. Further analysis has highlighted two major connections between the P + 1 loop and the catalytic loop (Arg165-Asn171). One emphasizes the hydrophobic packing of Tyr204 and Leu167 mediated through residues from the alphaF-helix, recently recognized as a signal integration motif, which together with the alphaE-helix forms the center of the hydrophobic core network. The other connection is mediated by the hydrogen bond interaction between Thr201 and Asp166, in a substrate-dependent manner. We speculate that the latter interaction may be important for the kinase to sense the presence of substrate and prepare itself for the catalytic reaction. Thus, the P + 1 loop is not merely involved in substrate binding; it mediates the communication between substrate and catalytic residues. (C) 2004 Elsevier Ltd. All rights reserved.