2GNI

PKA fivefold mutant model of Rho-kinase with inhibitor Fasudil (HA1077)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.27 Å
  • R-Value Free: 0.268 
  • R-Value Work: 0.188 
  • R-Value Observed: 0.192 

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This is version 1.3 of the entry. See complete history


Literature

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

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

(2006) J Biol Chem 281: 24818-24830

  • DOI: https://doi.org/10.1074/jbc.M512374200
  • Primary Citation of Related Structures:  
    2GFC, 2GNF, 2GNG, 2GNH, 2GNI, 2GNJ, 2GNL

  • PubMed 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.


  • Organizational Affiliation

    Group of Structural Biochemistry, German Cancer Research Center, 69120 Heidelberg.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
cAMP-dependent protein kinase, alpha-catalytic subunit350Bos taurusMutation(s): 8 
Gene Names: PRKACA
EC: 2.7.11.11
UniProt
Find proteins for P00517 (Bos taurus)
Explore P00517 
Go to UniProtKB:  P00517
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00517
Sequence Annotations
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  • Reference Sequence

Find similar proteins by:  Sequence   |   3D Structure  

Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
cAMP-dependent protein kinase inhibitor alphaB [auth I]20N/AMutation(s): 0 
UniProt & NIH Common Fund Data Resources
Find proteins for P61925 (Homo sapiens)
Explore P61925 
Go to UniProtKB:  P61925
PHAROS:  P61925
GTEx:  ENSG00000171033 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP61925
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
M77
Query on M77

Download Ideal Coordinates CCD File 
C [auth A]5-(1,4-DIAZEPAN-1-SULFONYL)ISOQUINOLINE
C14 H17 N3 O2 S
NGOGFTYYXHNFQH-UHFFFAOYSA-N
Modified Residues  2 Unique
IDChains TypeFormula2D DiagramParent
SEP
Query on SEP
A
L-PEPTIDE LINKINGC3 H8 N O6 PSER
TPO
Query on TPO
A
L-PEPTIDE LINKINGC4 H10 N O6 PTHR
Binding Affinity Annotations 
IDSourceBinding Affinity
M77 BindingDB:  2GNI Ki: min: 460, max: 1600 (nM) from 2 assay(s)
IC50: min: 541, max: 7605 (nM) from 12 assay(s)
Binding MOAD:  2GNI IC50: 2280 (nM) from 1 assay(s)
PDBBind:  2GNI IC50: 2280 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.27 Å
  • R-Value Free: 0.268 
  • R-Value Work: 0.188 
  • R-Value Observed: 0.192 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 72.227α = 90
b = 75.959β = 90
c = 80.396γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
CCP4data scaling
AMoREphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2006-05-23
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-11-10
    Changes: Database references, Derived calculations, Structure summary