Document Detail


Crystal structure of a cAMP-dependent protein kinase mutant at 1.26A: new insights into the catalytic mechanism.
MedLine Citation:
PMID:  14757059     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The catalytic subunit of cAMP-dependent protein kinase has served as a paradigm for the entire kinase family. In the course of studying the structure-function relationship of the P+1 loop (Leu198-Leu205) of the kinase, we have solved the crystal structure of the Tyr204 to Ala mutant in complexes with Mg.ATP and an inhibitory peptide at 1.26A, with overall structure very similar to that of the wild-type protein. However, at the nucleotide binding site, ATP was found largely hydrolyzed, with the products ADP-PO(4) retained in the structure. High-resolution refinement suggests that 26% of the molecules contain the intact ATP, whereas 74% have the hydrolyzed products. The observation of the substrate and product states in the same structure adds significant information to our understanding of the phosphoryl transfer process. Structural examination of the mutation site substantiates and extends the emerging concept that the hydrophobic core in the large lobe of the kinase might serve as a stable platform for anchoring key segments involved in catalysis. We propose that Tyr204 is critical for anchoring the P+1 loop to the core. Further analysis has highlighted two major connections between the P+1 loop and the catalytic loop (Arg165-Asn171). One emphasizes the hydrophobic packing of Tyr204 and Leu167 mediated through residues from the alphaF-helix, recently recognized as a signal integration motif, which together with the alphaE-helix forms the center of the hydrophobic core network. The other connection is mediated by the hydrogen bond interaction between Thr201 and Asp166, in a substrate-dependent manner. We speculate that the latter interaction may be important for the kinase to sense the presence of substrate and prepare itself for the catalytic reaction. Thus, the P+1 loop is not merely involved in substrate binding; it mediates the communication between substrate and catalytic residues.
Authors:
Jie Yang; Lynn F Ten Eyck; Nguyen Huu Xuong; Susan S Taylor
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of molecular biology     Volume:  336     ISSN:  0022-2836     ISO Abbreviation:  J. Mol. Biol.     Publication Date:  2004 Feb 
Date Detail:
Created Date:  2004-02-03     Completed Date:  2004-03-03     Revised Date:  2013-06-04    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  England    
Other Details:
Languages:  eng     Pagination:  473-87     Citation Subset:  IM    
Affiliation:
Howard Hughes Medical Institute, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Data Bank Information
Bank Name/Acc. No.:
PDB/1RDQ
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphatases / antagonists & inhibitors,  chemistry,  genetics,  metabolism
Adenosine Triphosphate / metabolism
Alanine / genetics,  metabolism
Amino Acid Sequence
Animals
Aspartic Acid / genetics,  metabolism
Binding Sites
Catalysis
Crystallography, X-Ray
Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors,  chemistry*,  genetics*,  metabolism
Hydrolysis
Hydrophobic and Hydrophilic Interactions
Mice
Models, Molecular
Molecular Sequence Data
Mutation / genetics*
Peptide Fragments / chemistry,  pharmacology
Threonine / genetics,  metabolism
Tyrosine / genetics,  metabolism
Grant Support
ID/Acronym/Agency:
GM19301/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Peptide Fragments; 0/protein kinase inhibitor peptide (5-24); 55520-40-6/Tyrosine; 56-41-7/Alanine; 56-65-5/Adenosine Triphosphate; 56-84-8/Aspartic Acid; 72-19-5/Threonine; EC 2.7.11.11/Cyclic AMP-Dependent Protein Kinases; EC 3.6.1.-/Adenosine Triphosphatases

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