Crystal Structure of the Potent Natural Product Inhibitor Balanol in Complex with the Catalytic Subunit of cAMP-Dependent Protein Kinase

Abstract

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...

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

“…6). The finding that active site inhibitors lock the phosphorylation sites on the C-terminal tail in a phosphatase-resistant conformation is consistent with structural studies of PKA that showed that the C-terminal tail is highly ordered when inhibitor is bound and highly disordered in the apo structure (39,40). It is interesting to note that occupancy of the active site by ATP analogues such as Bis I expels the autoinhibitory pseudosubstrate from the substrate-binding cavity (32), an event that, in itself, increases the phosphatase sensitivity of PKC by 2 orders of magnitude (7).…”

Balanol Analogues Probe Specificity Determinants and the Conformational Malleability of the Cyclic 3‘,5‘-Adenosine Monophosphate-Dependent Protein Kinase Catalytic Subunit,

“…The A-ring occupies the adenine ring subsite, the B-ring occupies the ribose subsite, and the C-and D-rings mimic the triphosphates, but occupy a distinct space. These spaces are the A-ring subsite, B-ring subsite, and C-and D-ring subsite (11). ATP (black stick) was taken from the C:ATP:IP20 structure (PDB code 1ATP) (2) and balanol (shown as a ball-andstick model) from the C:Bal structure (PDB code 1BX6) (11).…”

“…These spaces are the A-ring subsite, B-ring subsite, and C-and D-ring subsite (11). ATP (black stick) was taken from the C:ATP:IP20 structure (PDB code 1ATP) (2) and balanol (shown as a ball-andstick model) from the C:Bal structure (PDB code 1BX6) (11). Atoms are colored by type: oxygen (red), nitrogen (blue), and carbon (black).…”

Cytotoxicity and Proteomics Analyses of OSU03013 in Lung Cancer

“…The structure of PKA from the X-ray crystal complex structure (pdb code: 1bx6) (ref. 19) of PKA and bananol, which is an inhibitor of PKA, was used for docking OSU03013. To add hydrogen atoms, the all-atom molecular modeling program, Amber, was used first.…”

“…Atom charges on phosphoserine SEP338 and phosphothreonine TPO197 were obtained from Amber as well. For docking calculation, a docking space of 22.5 Â 22.5 Â 22.5 Å 3 centered around the ligand binding site (19) was used to ensure sufficient space exploration for ligand docking. Most other docking parameters were set to Autodock default parameters.…”