PDBsum entry 1q24

Transferase/transferase inhibitor

PDB id: 1q24

Contents

Protein chains

337 a.a. *

20 a.a. *

Ligands

ATP

Metals

_MG

Waters ×132

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Mutants of protein kinase a that mimic the ATP-Binding site of protein kinase b (akt).

Authors

M.Gassel, C.B.Breitenlechner, P.Rüger, U.Jucknischke, T.Schneider, R.Huber, D.Bossemeyer, R.A.Engh.

Ref.

J Mol Biol, 2003, 329, 1021-1034. [DOI no: 10.1016/S0022-2836(03)00518-7]

PubMed id

12798691

Abstract

The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the alpha isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAalpha V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAalpha V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.

Figure 2.
Figure 2. Sim-weighted electron density (2mF[o] -dF[c], blue at 1s) and difference electron density (mF[o] -dF[c], white at 2s and red at -2s) maps showing the rotation of Q181. The maps were calculated from a model of PKA (orange sticks) after refinement but prior to rotation of the side-chain. Green sticks depict the refined structure of the double mutant PKAB2. The density shows unambiguously how Q181 rotates to occupy the cavity near the V123A position and near the adenine-binding interaction sites.

Figure 5.
Figure 5. Stereo view of an overlay of the MgATP-PKAB2 complex structure (multicolored by atom type) and the MnAMP-PNP-PKA complex structure (green sticks, PDB code 1CDK[16.]). The 2F[o] -F[c] density (1s) belongs to PKAB2 liganded with ATP. The PKAB2 structure is the first Mg ATP-PKA structure and shows only a single bound metal ion.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 329, 1021-1034) copyright 2003.

Secondary reference #1

Title

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).

Authors

D.Bossemeyer, R.A.Engh, V.Kinzel, H.Ponstingl, R.Huber.

Ref.

Embo J, 1993, 12, 849-859.

PubMed id

8384554