Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT

Abstract

To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca(2+) ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca(2+) cations with Mg(2+) ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

Keywords: crystal structure; enzyme mechanism; molecular dynamics; phosphoryl transfer; protein kinase.

FIGURE 1.

A, electron density map for the active site components in PKAc-Ca₂ATP-CP20 contoured at 1.5σ level (4σ for calcium cations). B, electron density map for the active site components in PKAc-Ca₂AMPPNP-SP20 contoured at 1.5σ level (4σ for calcium cations). C, superposition of the active sites in PKAc-Ca₂ATP-CP20 (colored by atom type, carbon is green, Ca²⁺ ions are dark cyan, H₂O molecules red) and PKAc-Ca₂AMPPNP-SP20 (light magenta, carbon atoms; light cyan, Ca²⁺ ions; magenta, H₂O molecules), showing metals Ca1 and Ca2 bound at sites M1 and M2, respectively, nucleotides ATP and AMPPNP, Cys-21_CP20 and Ser-21_SP20 of the substrate peptides CP20 (blue carbon atoms) and SP20 (orange carbon atoms), respectively, and the residues of the enzyme that are important for metal binding or catalysis. Metal coordination, as black solid lines, and possible hydrogen bonds, as dashed lines, are shown for PKAc-Ca₂ATP-CP20. Distances are in Å. D, superposition of the active sites in pseudo-Michaelis complexes PKAc-Mg₂ATP-IP20 (PDB: 4DH3, dark magenta for all atoms including Mg²⁺ ions and water molecules), and PKAc-Ca₂ATP-CP20 (carbon, green; Ca²⁺ ions, dark cyan; and water molecules, red). Metal coordination are shownn as black solid lines, and possible hydrogen bonds as dashed blue are shown for PKAc-Mg₂ATP-IP20. Distances are in Å.

FIGURE 2.

Superposition of the active sites in pseudo-Michaelis complex PKAc-Ca₂ATP-CP20 (carbon are colored green, Ca²⁺ ions are dark cyan), transition state mimic PKAc-Mg₂ADP-MgF₃-SP20 (PDB code 1L3R, light pink, carbon atoms; magenta; Mg²⁺ ions, ball and stick model represents the MgF₃⁻ anion), and product complex PKAc-Ca₂ADP-pSP20 (blue, carbon atoms; light cyan, Ca²⁺ ions), showing similar conformation for the side chains of Ser-21_SP20 and Cys-21_CP20, both facing Asp-166, whereas in product complex C_β-O_γ is rotated away from Asp-166, suggesting the flip of the P-site residue following phosphoryl transfer. The distance between the γ-P of ATP and the oxygen in Ser-21_SP20 is shown as black dashed double arrow. The red dashed arrow demonstrates the difference in the position of γ-PO₃ group before and after the reaction. Distances are in Å.

FIGURE 3.

Superposition of the glycine-rich loop and the P-site residue of the substrate in pseudo-Michaelis complex PKAc-Ca₂ATP-CP20 (carbon colored green), transition state mimic PKAc-Mg₂ADP-MgF₃-SP20 (PDB ID 1L3R, light pink carbon atoms), and product complex PKAc-Ca₂ADP-pSP20 (PDB code 4IAK, blue carbon atoms) showing different conformations of the glycine-rich loop and positions of P-site side chain residue (red and black dashed arrows) relatively to the position of the glycine-rich loop at different stages of the phosphoryl transfer reaction.

FIGURE 4.

A, F_o − F_c omit difference electron density map for ADP, free phosphate and Cys-21_CP20 in two alternate conformations in PKAc-Mg₂ADP-PO₄-CP20 showing no density for the γ-phosphate of nucleotide. B, a close-up view of the enzyme active site in the PKAc-Mg₂ADP-PO₄-CP20 ternary complex showing metals Mg1 and Mg2 bound at sites M1 and M2, respectively, ADP, free phosphate, Cys-21_CP20 of pseudo-substrate peptide in two alternate conformations (blue carbon atoms), 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 blue dashed lines. Distances are in Å.

FIGURE 5.

A, r.m.s. deviation of the PKA catalytic domain backbone for PKAc-Ca₂ATP-CP20 (red), PKAc-Ca₂AMPPNP-SP20 (blue), and PKAc-Ca₂ADP-pSP20 (PDB code 4IAK, black) systems is plotted as a function of time. The color scheme is the same for all the following figures. B, red line shows distribution of distances between the S atom of Cys-21_CP20 and closest O atom in the Asp-166 carboxyl in PKAc-Ca₂ATP-CP20 system. Blue line shows distribution of distances between the O atom in Ser-21_SP20 and closest O atom in Asp-166 carboxyl in PKAc-Ca₂AMPPNP-SP20 system. C, distributions of the radius of gyration calculated for PKAc-Ca₂ATP-CP20, PKAc-Ca₂AMPPNP-SP20, and PKAc-Ca₂ADP-pSP20 systems. D, the distributions of trajectory projections onto the first and second principal components for PKAc-Ca₂ATP-CP20, PKAc-Ca₂AMPPNP-SP20 and PKAc-Ca₂ADP-pSP20 systems.

FIGURE 6.

Superposition of the active sites in pseudo-Michaelis complex PKAc-Ca₂ATP-CP20 (carbon colored green; dark cyan, Ca²⁺ ions), complex with free phosphate anion PKAc-Mg₂ADP-PO₄-CP20 (yellow carbon atoms; magenta, Mg²⁺ ions), and product complex PKAc-Ca₂ADP-pSP20 (PDB code 4IAK, blue carbon atoms; light cyan, Ca²⁺ ions) showing interactions formed by the γ-phosphate and water molecule, W4, at the position of the O3 atom of the free phosphate, in pseudo-Michaelis (red W4) and the product (blue W4) complexes. Metal coordination is shown as solid lines, whereas possible hydrogen bonds are represented as dashed lines.

FIGURE 7.

Snapshots of the phosphoryl transfer reaction based on crystallographic structures. A close-up view of the enzyme active site in (I) pseudo-Michaelis complex PKAc-Ca₂ATP-CP20 (green, dark cyan Ca²⁺ ions), (II) transition state mimic PKAc-Mg₂ADP-MgF₃-SP20 (PDB code 1L3R, pink, magenta Mg²⁺ ions), (III) PKAc-Mg₂ADP-PO₄-CP20 ternary complex (yellow, dark magenta Mg²⁺ ions), and (IV) product complex PKAc-Ca₂ADP-pSP20 (PDB code 4IAK, cyan, cyan Ca²⁺ ions). For II–IV, superposition of the present step (colored by atom type as described above) with the structure of the preceding step is shown in blue lines. Water molecules are represented by red and blue spheres for the present and preceding steps, respectively. Metal coordination is shown as solid lines, whereas possible hydrogen bonds are represented as dashed lines.