Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor.
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 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 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.
Sica
Filomena
F
Dipartimento di Chimica, Università degli Studi di Napoli Federico II, Via Cynthia, 80126 Naples, Italy.
Di Fiore
Anna
A
Merlino
Antonello
A
Mazzarella
Lelio
L
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides; D004722:Endoribonucleases / Q000737:chemistry* / Q000502:physiology*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry; D013997:Time Factors
D004220:Disulfides; D020033:Protein Isoforms; D004722:Endoribonucleases; D012260:Ribonucleases; C032523:ribonuclease SPL; D012259:Ribonuclease, Pancreatic; D003545:Cysteine