Crystal Structure of a Polyhistidine-Tagged Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase Complexed with the Peptide Inhibitor PKI(5−24) and Adenosine^†

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H₆-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5−24) and adenosine, was determined at 2.2 Å resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The “active” enzyme adopts a “closed” conformation as found in rC:PKI(5−24) [Knighton et al. (1991a,b) Science 253, 407−414, 414−420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the γ-phosphate of ATP. The glycine-rich loop between β1 and β2 helps to anchor the phosphates while the ribose ring is buried beneath β-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in β-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of β-strand 1, the glycine-rich loop, and β-strand 2. The adenine ring is buried beneath β-strand 1 and the linker strand (120−127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50−55) in the ATP binding domain have a mean B-factor of 41.4 Ų. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166−171) and the Mg²⁺ positioning loop (residues 184−186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg·ATP and the peptide inhibitor. In this ternary complex the γ-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the γ-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the γ-subsite.