Structural insights into conformational flexibility ofcAMP-dependent protein kinase from the unliganded and liganded catalytic subunit.

摘要

Crystal structures of the catalytic (C) subunit of adenosine-3 ',5'-cyclic monophosphate (cAMP) dependent protein kinase (PKA) were solved to understand the mechanism of ligand binding and to characterize the conformational flexibility of the active site.; The apoenzyme structure (Apo) provides a snapshot of the first step of the catalytic cycle. Surprisingly, many active site residues are preformed prior to ligand binding. A stable hydrophobic core---the C helix, beta strand 6, E helix, and F helix---appears to provide a stable scaffold, which rigidifies these residues. The surface created in the active site is contoured for discriminating ligand binding. The core also provides a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding site. Three potential lines of communication are the D helix, F helix, and Phe 238, which lies in the loop between the F and G helices. The junction of the small lobe and the large lobe forms a hydrophobic, "greasy", patch that may lubricate the sheering motion associated with domain rotation.; Crystal structures of the C subunit complexed with three different balanol analogs were solved to further probe the conformational flexibility of the active site and to understand why these analogs inhibit PKA more potently than calcium- and phospholipid-dependent protein kinase (PKC). Balanol is a fungal metabolite that inhibits PKA, PKG, and PKC, closely related members of the AGC subfamily of protein kinases. These analogs illustrate the adaptability of the C subunit active site. The D-ring subsite is more amenable to modification whereas the A-ring subsite is less tolerant. The C-terminal tail and the B helix of PKA influence the difference in binding affinity of PKA and PKC to the balanol analogs. BD2, an analog that is more hydrophobic than balanol, should pass through cell membranes and may be utilized as a tool for in vivo inhibition of PKA activity. The crystal structures of the C subunit described here provide insight to the design of specific and potent inhibitors of PKA and highlight subsite differences between PKA and PKC.