Document Detail


Crystal structure of the E230Q mutant of cAMP-dependent protein kinase reveals an unexpected apoenzyme conformation and an extended N-terminal A helix.
MedLine Citation:
PMID:  16253959     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Jian Wu; Jie Yang; Natarajan Kannan; Madhusudan; Nguyen-Huu Xuong; Lynn F Ten Eyck; Susan S Taylor
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  14     ISSN:  0961-8368     ISO Abbreviation:  Protein Sci.     Publication Date:  2005 Nov 
Date Detail:
Created Date:  2005-10-28     Completed Date:  2005-12-19     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2871-9     Citation Subset:  IM    
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.
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphate / chemistry,  metabolism
Amino Acid Substitution
Apoenzymes / chemistry
Binding Sites
Crystallography, X-Ray
Cyclic AMP-Dependent Protein Kinases / chemistry*,  genetics,  metabolism
Glutamic Acid / chemistry*,  genetics
Glutamine / chemistry,  genetics
Models, Molecular*
Mutagenesis, Site-Directed
Protein Conformation
Protein Structure, Secondary
Grant Support
ID/Acronym/Agency:
GM 19301/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Apoenzymes; 56-65-5/Adenosine Triphosphate; 56-85-9/Glutamine; 56-86-0/Glutamic Acid; EC 2.7.11.11/Cyclic AMP-Dependent Protein Kinases
Comments/Corrections

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