Both the diversity and similarity of cAMP binding sites are demonstrated by this new type II regulatory subunit structure. The structure represents an intramolecular paradigm for the cooperative triad that links two cAMP binding sites through a domain interface to the catalytic subunit of cAMP-dependent protein kinase. The domain interface surface is created by the binding of only one cAMP molecule and is enabled by amino acid sequence variability within the peptide chain that tethers the two domains together.

Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar K i values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 Å resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.Protein kinases, of which the human genome is predicted to encode several thousand, reversibly phosphorylate diverse molecular targets and are critical enzymes in the initiation, regulation, and abrogation of both normal and abnormal cellular functions (reviewed in refs 1-6). These often exquisitely specific kinases play essential roles in apoptosis, cell proliferation, gene expression, glycogen metabolism, immune response, neurotransmission, oncogenesis, and secondary messenger signal transduction (7-18). This biological pervasiveness underscores the importance of more explicitly characterizing kinase activation and inhibition. Additionally, there is significant therapeutic value in achieving selective pharmacological control of members of this important class of enzymes, since unregulated or otherwise defective protein kinase activities to date have been implicated in asthma, cancer, cardiovascular disorders, central nervous system (CNS) 1 diseases, diabetes, human immunodeficiency virus (HIV) infections, inflammation, psoriasis, and rheumatoid arthritis (19)(20)(21)(22)(23)(24).To more explicitly characterize protein kinase activation and inhibition, cAMP-dependent protein kinase, the most completely characterized and mechanistically simplest protein kinase (25, reviewed in refs 26-31), is a logical singular choice for biochemical and structural research focusing on cellular phosphorylation events at the molecular level. The inactive cAPK holoenzyme consists of two regulatory (R) and two catalytic (C) subunits that dissociate in response to elevated levels of intracellular cAMP. The triply phosphorylated, active C subunit then phosphorylates serine or threonine residues of peptide substrates containing the consensus sequence Arg-Arg-X-Ser[Thr]-Hyd, where X is variable and Hyd is any hydrophobic residue (3,14,32,33). The C subunit of cAPK shares with other kinase family members a conserved cat...