PDBsum entry 2gnf

Transferase/transferase inhibitor

PDB id: 2gnf

Contents

Protein chains

341 a.a. *

20 a.a. *

Ligands

Y27

Waters ×160

* Residue conservation analysis

PDB id:

2gnf

Name:

Transferase/transferase inhibitor

Title:

Protein kinase a fivefold mutant model of rho-kinase with y-27632

Structure:

Camp-dependent protein kinase, alpha-catalytic subunit. Chain: a. Synonym: pka c-alpha. Engineered: yes. Mutation: yes. Camp-dependent protein kinase inhibitor alpha. Chain: i. Fragment: pki(residues 5-24). Synonym: pki-alpha, camp-dependent protein kinase inhibitor,

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Gene: prkaca. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Other_details: the peptide was chemically synthesized. The sequence of the peptide is naturally found in rabbit.

Biol. unit:

Dimer (from PQS)

Resolution:

2.28Å R-factor: 0.201 R-free: 0.258

Authors:

S.Bonn,S.Herrero,C.B.Breitenlechner,R.A.Engh,M.Gassel,D.Bossemeyer

Key ref:

S.Bonn et al. (2006). Structural analysis of protein kinase A mutants with Rho-kinase inhibitor specificity. J Biol Chem, 281, 24818-24830. PubMed id: 16699172 DOI: 10.1074/jbc.M512374200

Date:

10-Apr-06 Release date: 23-May-06

Protein chain

P00517  (KAPCA_BOVIN) -  cAMP-dependent protein kinase catalytic subunit alpha from Bos taurus

Seq: 351 a.a.

Struc: 341 a.a.*

Protein chain

P63248  (IPKA_MOUSE) -  cAMP-dependent protein kinase inhibitor alpha from Mus musculus

Seq: 76 a.a.

Struc: 20 a.a.

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chain A: E.C.2.7.11.11 - cAMP-dependent protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M512374200

J Biol Chem 281:24818-24830 (2006)

PubMed id: 16699172

Structural analysis of protein kinase A mutants with Rho-kinase inhibitor specificity.

S.Bonn, S.Herrero, C.B.Breitenlechner, A.Erlbruch, W.Lehmann, R.A.Engh, M.Gassel, D.Bossemeyer.

ABSTRACT

Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.

Selected figure(s)

Figure 1.
FIGURE 1. A, detail of the substitution positions in the ATP binding site of PKA. B, the low molecular weight inhibitors used in this study.

Figure 9.
FIGURE 9. A, superposition of PKAR5-1077 (blue carbons) and (1Q8W) PKAWT-HA1077 (gray carbon atoms). B, electron density map (2F[o] - F[c] contoured at 1.5 ) of the inhibitor binding pocket of PKAR5-1077.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 24818-24830) copyright 2006.

Figures were selected by the author.