4HPU

Crystal structure of the catalytic subunit of cAMP-dependent protein kinase displaying partial phosphoryl transfer of AMP-PNP onto a substrate peptide


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.55 Å
  • R-Value Free: 0.194 
  • R-Value Work: 0.172 
  • R-Value Observed: 0.173 

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This is version 1.5 of the entry. See complete history


Literature

Phosphoryl transfer by protein kinase a is captured in a crystal lattice.

Bastidas, A.C.Deal, M.S.Steichen, J.M.Guo, Y.Wu, J.Taylor, S.S.

(2013) J Am Chem Soc 135: 4788-4798

  • DOI: https://doi.org/10.1021/ja312237q
  • Primary Citation of Related Structures:  
    4HPT, 4HPU

  • PubMed Abstract: 

    The catalytic (C) subunit of cAMP-dependent protein kinase (PKA) is a serine/threonine kinase responsible for most of the effects of cAMP signaling, and PKA serves as a prototype for the entire kinase family. Despite multiple studies of PKA, the steps involved in phosphoryl transfer, the roles of the catalytically essential magnesium ions, and the processes that govern the rate-limiting step of ADP release are unresolved. Here we identified conditions that yielded slow phosphoryl transfer of the γ-phosphate from the generally nonhydrolyzable analog of ATP, adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), onto a substrate peptide within protein crystals. By trapping both products in the crystal lattice, we now have a complete resolution profile of all the catalytic steps. One crystal structure refined to 1.55 Å resolution shows two states of the protein with 55% displaying intact AMP-PNP and an unphosphorylated substrate and 45% displaying transfer of the γ-phosphate of AMP-PNP onto the substrate peptide yielding AMP-PN and a phosphorylated substrate. Another structure refined to 2.15 Å resolution displays complete phosphoryl transfer to the substrate. These structures, in addition to trapping both products in the crystal lattice, implicate one magnesium ion, previously termed Mg2, as the more stably bound ion. Following phosphoryl transfer, Mg2 recruits a water molecule to retain an octahedral coordination geometry suggesting the strong binding character of this magnesium ion, and Mg2 remains in the active site following complete phosphoryl transfer while Mg1 is expelled. Loss of Mg1 may thus be an important part of the rate-limiting step of ADP release.


  • Organizational Affiliation

    Department of Pharmacology, University of California, San Diego, California 92093, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
cAMP-dependent protein kinase catalytic subunit alphaA [auth E]350Mus musculusMutation(s): 0 
Gene Names: PrkacaPkaca
EC: 2.7.11.11
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
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  • Reference Sequence

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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
cAMP-dependent protein kinase inhibitor alphaB [auth I]20Mus musculusMutation(s): 2 
UniProt & NIH Common Fund Data Resources
Find proteins for P63248 (Mus musculus)
Explore P63248 
Go to UniProtKB:  P63248
IMPC:  MGI:104747
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP63248
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.55 Å
  • R-Value Free: 0.194 
  • R-Value Work: 0.172 
  • R-Value Observed: 0.173 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 57.69α = 90
b = 79.8β = 90
c = 98.14γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-03-20
    Type: Initial release
  • Version 1.1: 2013-03-27
    Changes: Database references
  • Version 1.2: 2013-04-03
    Changes: Other
  • Version 1.3: 2013-04-10
    Changes: Database references
  • Version 1.4: 2017-11-15
    Changes: Advisory, Refinement description
  • Version 1.5: 2024-11-06
    Changes: Data collection, Database references, Derived calculations, Structure summary