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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
Author(s) -
Langer Thomas,
Vogtherr Martin,
Elshorst Bettina,
Betz Marco,
Schieborr Ulrich,
Saxena Krishna,
Schwalbe Harald
Publication year - 2004
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200400129
Subject(s) - protein kinase a , protein subunit , chemistry , chemical shift , kinase , cyclin dependent kinase complex , biochemistry , c raf , mitogen activated protein kinase kinase , stereochemistry , gene
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.