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J. Biol. Chem., Vol. 279, Issue 35, 36753-36760, August 27, 2004

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Structure and Stability of the Non-covalent Swapped Dimer of Bovine Seminal Ribonuclease

AN ENZYME TAILORED TO EVADE RIBONUCLEASE PROTEIN INHIBITOR^*

Filomena Sica, Anna Di Fiore, Antonello Merlino¶, and Lelio Mazzarella||

From the Dipartimento di Chimica, Università degli Studi di Napoli "Federico II," Via Cynthia, 80126 Napoli, Italy, the Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 6, 80134 Napoli, Italy, and the ^¶Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via ponte don Melillo, 84084, Fisciano (Sa), Italy

A growing number of pancreatic-type ribonucleases (RNases) present cytotoxic activity against malignant cells. The cytoxicity of these enzymes is related to their resistance to the ribonuclease protein inhibitor (RI). In particular, bovine seminal ribonuclease (BS-RNase) is toxic to tumor cells both in vitro and in vivo. BS-RNase is a covalent dimer with two intersubunit disulfide bridges between Cys³¹ of one chain and Cys³² of the second and vice versa. The native enzyme is an equilibrium mixture of two isomers, MxM and M=M. In the former the two subunits swap their N-terminal helices. The cytotoxic action is a peculiar property of MxM. In the reducing environment of cytosol, M=M dissociates into monomers, which are strongly inhibited by RI, whereas MxM remains as a non-covalent dimer (NCD), which evades RI. We have solved the crystal structure of NCD, carboxyamidomethylated at residues Cys³¹ and Cys³² (NCD-CAM), in a complex with 2'-deoxycitidylyl(3'-5')-2'-deoxyadenosine. The molecule reveals a quaternary structural organization much closer to MxM than to other N-terminal-swapped non-covalent dimeric forms of RNases. Model building of the complexes between these non-covalent dimers and RI reveals that NCD-CAM is the only dimer equipped with a quaternary organization capable of interfering seriously with the binding of the inhibitor. Moreover, a detailed comparative structural analysis of the dimers has highlighted the residues, which are mostly important in driving the quaternary structure toward that found in NCD-CAM.

Received for publication, May 20, 2004 , and in revised form, June 8, 2004.

^* This work was supported by Ministero Dell' Istruzione, Università e Ricerca (Grant PRIN2002). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1TQ9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^|| To whom correspondence should be addressed: Dipartimento di Chimica Università di Napoli "Federico II," Complesso Universitario di Monte Sant' Angelo, Via Cynthia, 80126 Napoli, Italy. Fax: 39-081-674090; E-mail: lelio.mazzarella{at}unina.it.

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				D. Picone, A. Di Fiore, C. Ercole, M. Franzese, F. Sica, S. Tomaselli, and L. Mazzarella The Role of the Hinge Loop in Domain Swapping: THE SPECIAL CASE OF BOVINE SEMINAL RIBONUCLEASE J. Biol. Chem., April 8, 2005; 280(14): 13771 - 13778. [Abstract] [Full Text] [PDF]