Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine

Biochemistry. 1997 Apr 15;36(15):4438-48. doi: 10.1021/bi961947+.


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 A 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 gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-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 beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-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 A2. 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 x ATP and the peptide inhibitor. In this ternary complex the gamma-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 gamma-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 gamma-subsite.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / metabolism
  • Adenosine / chemistry*
  • Adenosine / genetics
  • Adenosine / metabolism
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Catalysis
  • Crystallization
  • Crystallography, X-Ray
  • Cyclic AMP-Dependent Protein Kinases / chemistry*
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Histidine / chemistry*
  • Mice
  • Molecular Sequence Data
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism
  • Peptides / chemistry*
  • Phosphates / metabolism
  • Protein Conformation
  • Protein Kinase Inhibitors*
  • Recombinant Proteins / chemistry
  • Ribose / metabolism


  • Peptide Fragments
  • Peptides
  • Phosphates
  • Protein Kinase Inhibitors
  • Recombinant Proteins
  • protein kinase inhibitor peptide (5-24)
  • polyhistidine
  • Histidine
  • Ribose
  • Cyclic AMP-Dependent Protein Kinases
  • Adenine
  • Adenosine