CAMP-DEPENDENT PROTEIN-KINASE - CRYSTALLOGRAPHIC INSIGHTS INTO SUBSTRATE RECOGNITION AND PHOSPHOTRANSFER

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined a t 2.2 and 2.25 Angstrom resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary co mplex contains the phosphorylated substrate peptide. These 2 structure s were refined to crystallographic R-factors of 17.5 and 18.1%, respec tively. In the ternary complex, the hydroxyl oxygen OG of the serine a t the P-site is 2.7 Angstrom from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of thi s invariant carboxylate with a peptide substrate, and supports the pre dicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the ser ine 2.7 Angstrom from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the pho sphate on the Ser interacts directly with the epsilon N of Lys 168, an other conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma -phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuon g NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexe s. A comparison of this binary complex structure with the recently sol ved structure of the ternary complex containing ATP and inhibitor pept ide also reveals that the phosphate atom traverses a distance of about 1.5 Angstrom following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues a re seen when the substrate and product complexes are compared, althoug h the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this str uctural motif is a highly mobile segment of the protein.