The catalytic subunit of cAMP-dependent protein kinase represents the basic catalytic subunit that is conserved in every protein kinase and therefore, owing to the structural homology between protein kinases, may serve as a model for viewing the entire family of over one hundred protein kinases. Crystallization studies resulted in several different complexes in tow different space groups (P212121 and P6122). Structure solution of the catalytic subunit will be based on our best crystal, an orthorhombic crystal (P2121) of a binary complex of recombinant catalytic subunit with a specific 20-amino acid peptide inhibitor. A mercury derivative was used to identify two cysteine residues, Cys 199 and Cys 343. The """"""""phasing power"""""""" (fr.m.s./Er.m.s.) of this derivative ranged from 3.13 to 1.91 within the resolution range of 10.15Angstrom to 3.61Angstrom. An electron density map to 2.7Angstrom resolution has been obtained by first generating a 3.5Angstrom resolution map using the B.C. Wang error filtering method and, second, resolution extension. The CdATP binding site was located using CdATP co-crystals of the ternary complex, with a specific peptide inhibitor. The identified ATP binding site appears in the immediate vicinity of Cys 199, as predicted from other biochemical studies. Within the elongated bilobal molecule, seven helices and nine beta-strands have been identified. Model building at the present stage consists of 90% alpha-carbons. Structure solution of the hexagonal crystal (P6122) of the pig heart catalytic subunit (myristic acid present) ternary complex with MgATP and a specific peptide inhibitor is based on the molecular envelope of the binary complex and the method of molecular replacement. The molecular orientation in the hexagonal unit cell was determined using the fast rotation function and data in the resolution range of 5.0Angstrom to 3.0Angstrom. The translational function applied to all Harker sections determined the molecular position and selected the correct space group. The real space orientation matrix is: 0.5331, -0.0356, 1.0000, 1.0504, - 0.0701 -0.0101, 0.0150, 0.2625, 0.0000 and the position of Hg-Cys 199 is: x= -0.082; y= -0.371; z= -0.033. This position corresponds to the position of Hg-Cys 199 x= 0.365, y= -0.133, z= 0.542 in the orthorhombic cell (P212121). Structure solution of two crystal forms will reveal not only the architecture of the catalytic subunit with and without myristic acid, but also the topology of the ATP and peptide binding sites. Furthermore, those structural studies will allow to proceed the crystallographic analysis of various mutants, the first of which has already been crystallized (Cys 343->Ser).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Diego
Schools of Arts and Sciences
La Jolla
United States
Zip Code
Sowadski, J M; Epstein, L F; Lankiewicz, L et al. (1999) Conformational diversity of catalytic cores of protein kinases. Pharmacol Ther 82:157-64
Sowadski, J M (1996) Introduction: crystallization of membrane proteins--in need of a new focus? J Bioenerg Biomembr 28:3-5
Sowadski, J M; Ellis, C A; Madhusudan (1996) Detergent binding to unmyristylated protein kinase A--structural implications for the role of myristate. J Bioenerg Biomembr 28:7-12
Vihinen, M; Vetrie, D; Maniar, H S et al. (1994) Structural basis for chromosome X-linked agammaglobulinemia: a tyrosine kinase disease. Proc Natl Acad Sci U S A 91:12803-7
Madhusudan; Trafny, E A; Xuong, N H et al. (1994) cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer. Protein Sci 3:176-87
Knighton, D R; Cadena, D L; Zheng, J et al. (1993) Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor. Proc Natl Acad Sci U S A 90:5001-5
Zheng, J; Knighton, D R; Xuong, N H et al. (1993) Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations. Protein Sci 2:1559-73
Gibbs, C S; Knighton, D R; Sowadski, J M et al. (1992) Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit. J Biol Chem 267:4806-14