Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5-24).

Abstract

The crystal structure of the porcine heart catalytic subunit of cAMP‐dependent protein kinase in a ternary complex with the MgATP analogue MnAMP‐PNP and a pseudosubstrate inhibitor peptide, PKI(5‐24), has been solved at 2.0 A resolution from monoclinic crystals of the catalytic subunit isoform CA. The refinement is presently at an R factor of 0.194 and the active site of the molecule is well defined. The glycine‐rich phosphate anchor of the nucleotide binding fold motif of the protein kinase is a beta ribbon acting as a flap with conformational flexibility over the triphosphate group. The glycines seem to be conserved to avoid steric clash with ATP. The known synergistic effects of substrate binding can be explained by hydrogen bonds present only in the ternary complex. Implications for the kinetic scheme of binding order are discussed. The structure is assumed to represent a phosphotransfer competent conformation. The invariant conserved residue Asp166 is proposed to be the catalytic base and Lys168 to stabilize the transition state. In some tyrosine kinases Lys168 is functionally replaced by an Arg displaced by two residues in the primary sequence, suggesting invariance in three‐dimensional space. The structure supports an in‐line transfer with a pentacoordinate transition state at the phosphorus with very few nuclear movements.

Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

“…Usually, such non-proline cis-peptides are found close to the active site or catalytic center of an enzyme. This is obviously the case here, because the glycinerich loop frames the border of the active site and participates in binding and orienting the triphosphoryl group of ATP (33). These new glycine flap orientations, especially those of groups two and three, have not been observed previously.…”

“…It binds not only to the 3ЈOH-group of ATP but has an important role in recognition of the substrate consensus sequence of PKA (33,34). Glu-127 interacts with Arg-2 (Arg-18 in PKI(5-24)) of the substrate recognition sequence RRX(S/ T)Y. Asp-127, conserved in most AGC kinases, and in many others, is a true homologue of Glu-127 in this respect, because it makes a bidentate contact to the guanidinium group of Arg-18 from the bound PKI(5-24) pseudosubstrate peptide.…”

Enzyme‐mononucleotide interactions: Three different folds share common structural elements for atp recognition

“…The star-stop positions of the secondary structures are noted at the right of the segments according to the X-ray crystallographic data (Bossemeyer et al, 1993;Eriksson et al, 1997;Fan et al, 1997). Positions of equivalent residues forming hydrogen bonds and hydrophobic interactions with the cofactors are marked by "*" and ''n ," respectively.…”

“…These last two fold classes are particularly interesting. Although the folds are different, two of their typical members, CAMP-dependent protein kinase (cAPK) from the one family (Bossemeyer et al, 1993) and D-Ala:D-Ala ligase (m-ligase) from the other family (Fan et al, 1994(Fan et al, , 1997, have over 100 structurally-equivalent residues from ten segments that form two identical supersecondary structures between which the cofactor ATP is bound in a similar way (Denessiouk et al, 1998).…”

Structural basis for the impact of phosphorylation on the activation of plant receptor-like kinase BAK1

“…A similar observation on the lack of interaction between the bound ATP or AMP-PNP molecule and the P-loop was reported in the crystal structure of insulin receptor tyrosine kinase (IRK) [10] (Supplementary information, Figure S6D). By contrast, the P-loops of the cAMP-dependent protein kinase (cAPK) (Supplementary information, Figure S6B) and the γ-subunit of phosphorylase kinase (PHK) (Supplementary information, Figure S6C) significantly contribute to the stabilization of the β-and γ-phosphates of ATP [11]. This phenomenon indicates the variations in the stabilization of ATP binding by the P-loop of different kinase family members.…”