The final model shows clear density for the C subunit except for the first 10 residues. loop is usually oriented parallel to the phenyl ring of N46, forming a strong -stacking conversation, whereas the analogous Phe-54 in PKA C rotates 30 and forms a weaker conversation. Structural comparison revealed that steric hindrance between the preceding Ser-53 and the propoxy group of the phenyl ring may explain the weaker conversation with PKA C. The analogous Gly-370 in PKG I, however, causes little steric hindrance with Phe-371. Moreover, Ile-406 around the C helix forms a hydrophobic conversation with N46 whereas its counterpart in PKA, Thr-88, does not. Substituting these residues in MKC9989 PKG I with those in PKA C increases the IC50 values for N46, whereas replacing these residues in PKA C with those in PKG I reduces the IC50, consistent with our structural findings. In conclusion, our results explain the structural basis for N46-mediated selective inhibition of human PKG I and provide a starting point for structure-guided design of selective PKG I inhibitors. and labeled with the corresponding residue numbers. Phosphorylated residues are indicated (PKG I T517 and PKA C S139/T197/S338). and and fluorine, omit maps of N46 (contoured at 3.0 level). Three classes of small-molecule PKG inhibitors have been widely used for functional studies of PKG (23, 24). The first class is the R-diastereomer of the phosphorothioate analogs of cGMP, including Rp-cGMPS (25). This compound binds the R-domain and stabilizes its inactive state without causing conformational changes required for activation (26). The second class consists of small molecules that compete with ATP by directly binding the active site within the C-domain. These reagents include H-89, balanol, and KT-5823 (27,C32). The third class includes peptide inhibitors that also bind the active site and prevent substrate binding. However, all of these inhibitors lack potency, specificity, and activity = 20 m) and nonselectively inhibits other cyclic nucleotide effectors, such as phosphodiesterase and PKA (23). KT-5823 also inhibits other kinases and may not inhibit PKG in intact cells (33). Despite its high potency and Fig. S1). The final model shows clear density for the C subunit except for the first 10 residues. Unlike previous PKA C structures, the N-terminal A helix disengages from the catalytic core because of unusual crystal packing interactions (Fig. Rabbit polyclonal to ZNF703.Zinc-finger proteins contain DNA-binding domains and have a wide variety of functions, most ofwhich encompass some form of transcriptional activation or repression. ZNF703 (zinc fingerprotein 703) is a 590 amino acid nuclear protein that contains one C2H2-type zinc finger and isthought to play a role in transcriptional regulation. Multiple isoforms of ZNF703 exist due toalternative splicing events. The gene encoding ZNF703 maps to human chromosome 8, whichconsists of nearly 146 million base pairs, houses more than 800 genes and is associated with avariety of diseases and malignancies. Schizophrenia, bipolar disorder, Trisomy 8, Pfeiffer syndrome,congenital hypothyroidism, Waardenburg syndrome and some leukemias and lymphomas arethought to occur as a result of defects in specific genes that map to chromosome 8 S2). The A helix of a neighboring symmetry mate occupies the equivalent position seen in previous structures and provides the same set of interactions with the catalytic core. The overall structure of the PKG I CCN46 complex is similar to the AMP-PNP-bound structure.4 It shows a closed conformation with the fully ordered glycine-rich loop and C-terminal tail (Fig. 1Residues contacting N46 are shown as Hydrogen bonds are shown as indicate key VWD interactions, with distances indicated in angstroms. The A-ring (indazole ring) binds the adenine MKC9989 subsite consisting of the hinge (loop between 5-D) and hydrophobic residues from both small and large lobes (Fig. 2Hydrogen bonds are shown as and The rings of balanol and N46 are labeled and G370S and I406T). We also mutated these two PKA C residues into the corresponding MKC9989 PKG I residues (S53G and T88I). For PKG I, we generated two single mutants (G370S and I406T) and a double mutant (G370S/I406T). For PKA C, we only generated a double mutant (S53G/T88I). We then measured IC50 values using kinase assays (Fig. 5). N46 showed an IC50 of 43 nm for WT PKG I, whereas it inhibited PKA C with an IC50 of MKC9989 1030 nm, showing an 24-fold difference in selectivity. The PKG I single mutants were inhibited with higher IC50 values of 90 nm and 142 nm for G370S and I406T, respectively. The double mutant PKG I showed an IC50 value of 301 nm, demonstrating a synergistic MKC9989 effect of the two mutations. In contrast, the PKA C double mutant showed an IC50 of 552 nm, which is almost half of that.