Unexpected binding mode for 2′-phosphoadenosine-based nucleotide inhibitors in complex with human angiogenin revealed by heteronuclear NMR spectroscopy

Human angiogenin (Ang) is a tumor-promoting RNase in the pancreatic RNase superfamily. Efforts to develop nucleotide-based inhibitors of Ang as potential anticancer drugs have been hampered by the lack of direct structural information on Ang-nucleotide complexes. Here, we have used heteronuclear NMR spectroscopy with 15N- and 15N/13C-labeled Ang to map the interactions of Ang with the phosphate ion, seven adenosine mononucleotides (the 2′-, 3′-, and 5′-monophosphates, the 2′,5′- and 3′,5′-diphosphates, the 5′-diphosphate, and the 2′-monophospho-5′-diphosphate), and the dinucleotide 2′-deoxyuridine 3′-pyrophosphate (P′ → 5′) adenosine-2′-phosphate (dUppA-2′-p). The 2′-phosphate based derivatives, which bind more tightly than the corresponding 3′-phosphate isomers, induced characteristic large resonance perturbations of the backbone amide proton of Leu115, the backbone 15N of His 114, and the Gln12 side-chain NH2 group in the Ang active site. In contrast, adenosine derivatives with only 3′- or 5′-phosphates produced much less dramatic perturbations of Leu 115 and His114 resonances, along with modest perturbations of additional residues both within and beyond the active site. Measurements of NOEs together with molecular docking analyses revealed the three-dimensional structures of the complexes of Ang with adenosine 2′,5′-diphosphate and dUppA-2′-p; the binding modes of these inhibitors differ substantially from those predicted in earlier studies. Most notably, the 2′-phosphate rather than the 5′-phosphate occupies the P1 catalytic subsite of Ang, and the side chain of His114 has undergone a conformational transition that positions it outside P 1 and allows it to form stacking interactions with the adenine ring of the inhibitor. Strikingly, the 2′-deoxyuridine moiety of dUppA-2′-p makes only a few contacts with Ang, and these involve residues outside the B1 subsite where the pyrimidine ring of substrates normally binds.