In this study, we investigate the inhibition of human angiogenin by ammonium sulfate. The inhibitory potency of ammonium sulfate for human angiogenin (IC50 = 123.5 ± 14.9 mm) is comparable to that previously reported for RNase A (119.0 ± 6.5 mm) and RNase 2 (95.7 ± 9.3 mm). However, analysis of two X-ray crystal structures of human angiogenin in complex with sulfate anions (in acidic and basic pH environments, respectively) indicates an entirely distinct mechanism of inhibition. While ammonium sulfate inhibits the ribonucleolytic activity of RNase A and RNase 2 by binding to the active site of these enzymes, sulfate anions bind only to peripheral substrate anion-binding subsites of human angiogenin, and not to the active site.

PDB References: ribonuclease A, U5P complex, 3dxg, r3dxgsf; UDP complex, 3dxh, r3dxhsf. In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5 0 -phosphate (U5P) and uridine 5 0 -diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with K i values of 4.0 and 0.65 mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purinebinding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily.

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domain were grown under paraffin oil from a mixture of ammonium sulfate and alcohols. The space group is I4 1 32 with a=155.3 Å. The 2.5 Å data set was collected at the SER-CAT beamline 22-ID at the single wavelength of 1.283 Å. The crystal structure was solved by SAD approach utilizing anomalous diffraction signal of the bound Zn atoms and was refined with REFMAC to an R-factor of 20.1% (R-free=22.7%). The structure comprises a helical bundle held by three Zn fingers and is very similar to the solution structures determined for the shorter peptide [1, 2] corresponding to the evolutionarily conserved Taz2 domain from CBP and p300. Residues 1813-1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal contact interactions with the peptide binding site of Taz2. The structure thus provides information relevant to the specificity of CBP/p300 interactions with transcription factors.

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