Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase: an X-ray crystallographic study of complexes with various metals and peptide substrate SP20

Abstract

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg(2+), Ca(2+), Sr(2+), and Ba(2+) metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg(2+), nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5'-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues.

Figure 1

(a) Close-up view of the enzyme active site in the PKAc–Mg₂AMP-PCP–SP20 pseudo-Michaelis complex showing metals Mg1 and Mg2 bound at sites M1 and M2, respectively, AMP-PCP, Ser21_SP20, and the residues of the enzyme that are important for metal binding or catalysis. Metal coordination is shown as black solid lines, whereas possible hydrogen bonds are represented as red dashed lines. (b) Electron density for the active site components AMP-PCP, Mg²⁺, water molecules, and Ser21_SP20 of the substrate peptide SP20 contoured at the 2.0σ level.

Figure 2

Superposition of the active sites of PKAc–Mg₂AMP-PCP–SP20 (colored by atom type, carbon colored green), PKAc–Mg₂ADP–pSP20 (magenta), and PKAc–Mg₂ATP–IP20 (cyan). The distance between the γ-P atom of ATP and the nucleophilic oxygen of Ser21_SP20 is only 3.0 Å, indicating correct positioning of the reactants in the studied complexes. Distances are in angstroms.

Figure 3

Close-up view of the enzyme active site in the PKAc–Mg₂ADP–pSP20 product complex showing metals Mg1 and Mg2 bound at sites M1 and M2, respectively, ADP, phosphorylated pSer21_SP20, and the residues of the enzyme that are important for metal binding or catalysis. Metal coordination is shown as black solid lines, whereas possible hydrogen bonds are represented as red dashed lines.

Figure 4

Superposition of the active sites of PKAc–Mg₂ADP–pSP20 and PKAc–Mg₂ATP–IP20. The distance between the γ-P atom of ATP and the phosphorus in pSer21_SP20 is shown as a black dashed arrow, indicating that the phosphate has to move <2 Å during the phosphoryl transfer reaction to its new position in pSer21_SP20.

Figure 5

Electron density for the active site components ADP, pSer21_SP20, and Ser53 contoured at the 2.0σ level (4σ for calcium cations) in PKAc–Ca₂ADP–pSP20. Distances are in angstroms.