2GNI: Pka Fivefold Mutant Model Of Rho-Kinase With Inhibitor Fasudil (Ha1077)

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.
PDB ID: 2GNIDownload
MMDB ID: 39399
PDB Deposition Date: 2006/4/10
Updated in MMDB: 2012/12
Experimental Method:
x-ray diffraction
Resolution: 2.27  Å
Source Organism:
synthetic construct
Similar Structures:
Biological Unit for 2GNI: dimeric; determined by author and by software (PISA)
Molecular Components in 2GNI
Label Count Molecule
Proteins (2 molecules)
Camp-dependent Protein Kinase, Alpha-catalytic Subunit(Gene symbol: PRKACA)
Molecule annotation
Camp-dependent Protein Kinase Inhibitor Alpha(Gene symbol: PKIA)
Molecule annotation
Chemical (1 molecule)
* Click molecule labels to explore molecular sequence information.

Citing MMDB