NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase.
Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.
Johann Wolfgang Goethe-Universität Frankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Marie Curie Strasse 11, 60439 Frankfurt am Main, Germany. t.langer@nmr.uni-frankfurt.de
Thomas
T
Langer
Martin
M
Vogtherr
Bettina
B
Elshorst
Marco
M
Betz
Ulrich
U
Schieborr
Krishna
K
Saxena
Harald
H
Schwalbe
D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D013329:Structure-Activity Relationship
D021122:Protein Subunits; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases