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

Abstract

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H 6 -rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heatstable 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 1 and 2 helps to anchor the phosphates while the ribose ring is buried beneath -strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in -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 -strand 1, the glycine-rich loop, and -strand 2. The adenine ring is buried beneath -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 glycinerich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 Å 2 . 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 Mg 2+ 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.

Crystal Structures of Interleukin-2 Tyrosine Kinase and Their Implications for the Design of Selective Inhibitors

“…deviation for 245 C-␣ atoms of 0.37 Å. In both structures, the ␣C-helix is completely ordered, placing the catalytically important residues Glu 406 , Lys 391 , and Asp 500 in an orientation that resembles the active form of a protein kinase (by reference to the structure of cAMP-dependent protein kinase (22)). …”

Structural insights into mis-regulation of protein kinase A in human tumors

“…1, Table 1, and Table S1): the full-length wild-type C subunit (PKAc), the predominant point mutant (PKAc L205R), the predominant chimeric fusion protein (DnaJ-PKAc), and PKAc with exon 1 residues deleted (PKAc Δexon1). All structures were determined as complexes with ATP and the protein kinase inhibitor (PKI) peptide (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In each, ATP is bound with two metal ions within the cleft formed between the smaller N-terminal and larger C-terminal lobes of the C subunit, associated as previously described (14).…”

“…2 A and B), it is immediately clear that PKI is bound differently in the vicinity of R205. In the structure of PKAc, binding of PKI is as seen before (16) in an extended conformation along the active-site cleft with the sidechain of A21 (P0) pointing toward the γ-phosphate of ATP located 3.7 Å away. Protein-peptide contacts include ionic interactions between PKI arginine side-chains at positions 15 (P−6), 18 (P−3), and 19 (P−2) with a cluster of PKAc glutamate sidechains (positions 170, 203, 230), van der Waals contacts between the side-chains of PKI H23 (P+2) and PKAc F187 and L82, and a hydrogen bond between the side-chains of the C-terminal D24 (P+3) of PKI and K83 of PKAc.…”

Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

“…The glycine-rich loop in protein kinases is a characteristic feature of the enzyme active site, and interactions between amino acids in this loop and bound nucleotide appear to be crucial for protein kinase function on the basis of the available C-subunit crystal structures (14,20,21). Although perturbations in steady-state kinetic parameters were measured previously for mutations of the C-subunit of cAPK (23) that were specifically perturbed at the three glycines in the glycine-rich loop, effects on substrate binding, phosphoryl transfer, and net product release were not determined.…”

“…Figure 4d compares crystallographic B-factors from structures with and without ATP. While Gly52 and Ser53 are highly flexible in the binary complex that lacks ATP, these amino acids are ordered in the ternary complex when both ATP and the inhibitor peptide are present (14).…”