Crystal structure of RNaseA tandem enzymes and their interaction with the cytosolic ribonuclease inhibitor

• Published in: The FEBS journal Vol. 278; no. 2; p. 331
• Main Authors: Arnold, Ulrich, Leich, Franziska, Neumann, Piotr, Lilie, Hauke, Ulbrich-Hofmann, Renate
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell Publishing Ltd 01.01.2011
• Subjects: Binding sites; Biochemistry; Crystal structure; Enzymes; Molecular biology
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2010.07957.x

Because of their ability to degrade RNA, RNases are potent cytotoxins. The cytotoxic activity of most members of the RNaseA superfamily, however, is abolished by the cytosolic ribonuclease inhibitor (RI). RNaseA tandem enzymes, in which two RNaseA molecules are artificially connected by a peptide linker, and thus have a pseudodimeric structure, exhibit remarkable cytotoxic activity. Invitro, however, these enzymes are still inhibited by RI. Here, we present the crystal structures of three tandem enzymes with the linker sequences GPPG, SGSGSG, and SGRSGRSG, which allowed us to analyze the mode of binding of RI to the RNaseA tandem enzymes. Modeling studies with the crystal structures of the RI-RNaseA complex and the SGRSGRSG-RNaseA tandem enzyme as templates suggested a 1:1 binding stoichiometry for the RI-RNaseA tandem enzyme complex, with binding of the RI molecule to the N-terminal RNaseA entity. These results were experimentally verified by analytical ultracentrifugation, quantitative electrophoresis, and proteolysis studies with trypsin. As other dimeric RNases, which are comparably cytotoxic, either evade RI binding or potentially even bind two RI molecules, inactivation by RI cannot be the crucial limitation to the cytotoxicity of dimeric RNases. [PUBLICATION ABSTRACT]