Document Detail

Phosphoryl transfer by protein kinase A is captured in a crystal lattice.
MedLine Citation:
PMID:  23458248     Owner:  NLM     Status:  MEDLINE    
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.
Adam C Bastidas; Michael S Deal; Jon M Steichen; Yurong Guo; Jian Wu; Susan S Taylor
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-03-12
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  135     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-03-27     Completed Date:  2013-09-09     Revised Date:  2013-10-17    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4788-98     Citation Subset:  IM    
Department of Pharmacology, University of California, San Diego, California 92093, USA.
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MeSH Terms
Adenylyl Imidodiphosphate / chemistry,  metabolism*
Crystallography, X-Ray
Cyclic AMP-Dependent Protein Kinases / chemistry,  metabolism*
Magnesium / chemistry,  metabolism
Models, Molecular
Peptides / chemistry,  metabolism*
Grant Support
F31 GM099415/GM/NIGMS NIH HHS; F31GM099415/GM/NIGMS NIH HHS; GM19301/GM/NIGMS NIH HHS; R01 GM019301/GM/NIGMS NIH HHS; T32 CA009523/CA/NCI NIH HHS; T32 GM007752/GM/NIGMS NIH HHS; //Howard Hughes Medical Institute; //Howard Hughes Medical Institute
Reg. No./Substance:
0/Peptides; 25612-73-1/Adenylyl Imidodiphosphate; 7439-95-4/Magnesium; EC AMP-Dependent Protein Kinases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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