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 ca talytic subunit (H-6-rC) of cAMP-dependent protein kinase (cAPK), comp lexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 Angst rom resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final cr ystallographic R-factor of 18.2% with good stereochemical parameters. The ''active'' enzyme adopts a ''closed'' conformation as found in rC: PK1(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] an d packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nu cleotide binding site found in the protein kinase family. The adenosin e occupies a mostly hydrophobic pocket at the base of the cleft betwee n the two lobes and is completely buried. The missing triphosphate moi ety of ATP is filled with a water molecule (Wtr 415) which replaces th e gamma-phosphate of ATP. The glycine-rich loop between beta 1 and bet a 2 helps to anchor the phosphates while the ribose ring is buried ben eath beta-strand 2. Another ordered water molecule (Wtr 375) is pentac oordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, G lu 127 in the linker strand between the two lobes, Tyr 330, and a thir d water molecule, Wtr 359. The conserved nucleotide fold can be define d as a lid comprised of P-strand 1, the glycine-rich loop, and P-stran d 2. The adenine ring is buried beneath beta-strand 1 and the linker s trand (120-127) that joins the small and large lobes. The C-terminal t ail containing Tyr 330, a segment that lies outside the conserved core , covers this fold and anchors it in a closed conformation. The main-c hain atoms of the flexible glycine-rich loop (residues 50-55) in the A TP binding domain have a mean B-factor of 41.4 Angstrom(2). This loop is quite mobile, in striking contrast to the other conserved loops tha t converge at the active site cleft. The catalytic loop (residues 166- 171) and the Mg2+ positioning loop (residues 184-186) are a stable par t 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 t he ligands that occupy the active site cleft with maximum stability ac hieved in the ternary complex containing Mg ATP and the peptide inhibi tor, In this ternary complex the gamma-phosphate is secured between bo th lobes by hydrogen bonds to the backbone amide of Ser 53 in the glyc ine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma- phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no cont act with the small lobe. This glycine-rich loop is thus the most mobil e component of the active site cleft, with the tip of the loop being h ighly sensitive to what occupies the gamma-subsite.