1SYK

Crystal structure of E230Q mutant of cAMP-dependent protein kinase reveals unexpected apoenzyme conformation


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.80 Å
  • R-Value Free: 0.249 
  • R-Value Work: 0.202 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix.

Wu, J.Yang, J.Kannan, N.Madhusudan, N.Xuong, N.H.Ten Eyck, L.F.Taylor, S.S.

(2005) Protein Sci 14: 2871-2879

  • DOI: https://doi.org/10.1110/ps.051715205
  • Primary Citation of Related Structures:  
    1SYK

  • PubMed Abstract: 

    Glu230, one of the acidic residues that cluster around the active site of the catalytic subunit of cAMP-dependent protein kinase, plays an important role in substrate recognition. Specifically, its side chain forms a direct salt-bridge interaction with the substrate's P-2 Arg. Previous studies showed that mutation of Glu230 to Gln (E230Q) caused significant decreases not only in substrate binding but also in the rate of phosphoryl transfer. To better understand the importance of Glu230 for structure and function, we solved the crystal structure of the E230Q mutant at 2.8 A resolution. Surprisingly, the mutant preferred an open conformation with no bound ligands observed, even though the crystals were grown in the presence of MgATP and the inhibitor peptide, IP20. This is in contrast to the wild-type protein that, under the same conditions, prefers the closed conformation of a ternary complex. The structure highlights the importance of the electrostatic surface not only for substrate binding and catalysis, but also for the mechanism for closing the active site cleft. This surface mutation clearly disrupts the recognition and binding of substrate peptide so that the enzyme prefers an open conformation that cannot trap ATP. This is consistent with the reinforcing concepts of conformational dynamics and the synergistic binding of ATP and substrate peptide. Another unusual feature of the structure is the observation of the entire N terminus (Gly1-Thr32) assumes an extended alpha-helix conformation. Finally, based on temperature factors, this mutant structure is more stable than the wild-type C-subunit in the apo state.


  • Organizational Affiliation

    Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, Leichtag 415, La Jolla, CA 92093, USA.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
cAMP-dependent protein kinase, alpha-catalytic subunit
A, B
350Mus musculusMutation(s): 3 
Gene Names: PRKACAPKACA
EC: 2.7.1.37
UniProt
Find proteins for P05132 (Mus musculus)
Explore P05132 
Go to UniProtKB:  P05132
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP05132
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  2 Unique
IDChains TypeFormula2D DiagramParent
SEP
Query on SEP
A, B
L-PEPTIDE LINKINGC3 H8 N O6 PSER
TPO
Query on TPO
A, B
L-PEPTIDE LINKINGC4 H10 N O6 PTHR
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.80 Å
  • R-Value Free: 0.249 
  • R-Value Work: 0.202 
  • Space Group: P 42
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 120.3α = 90
b = 120.3β = 90
c = 58.7γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
CNSrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-05-17
    Type: Initial release
  • Version 1.1: 2008-04-30
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-02-01
    Changes: Structure summary
  • Version 1.4: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.5: 2023-08-23
    Changes: Data collection, Refinement description