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 Cys 31 of one chain and Cys 32 of the second and vice versa. The native enzyme is an equilibrium

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... ture 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 Cys 31 and Cys 32 (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. 1 The abbreviations used are: BS-RNase, bovine seminal ribonuclease; MxM, dimeric form of BS-RNase in which the chains swap their N-terminal tails; MϭM, unswapped dimer of BS-RNase; NCD, non-covalent dimericswapped form of BS-RNase; NCD-CAM, S-[Cys 31 ]carboxyamidomethyl, S-[Cys 32 ]carboxyamidomethyl-NCD; RNase A, bovine pancreatic ribonuclease; dCpdA, 2Ј-deoxycitidylyl(3Ј-5Ј)-2Јdeoxyadenosine; PDB, Protein Data Bank; r.m.s.d., root mean square deviation; RI, ribonuclease protein inhibitor; MCAM, monomeric carboxyamidomethylated derivative of BS-RNase.