The buried diversity of bovine seminal ribonuclease: shape and cytotoxicity of the swapped non-covalent form of the enzyme |
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Authors: | Merlino Antonello Ercole Carmine Picone Delia Pizzo Elio Mazzarella Lelio Sica Filomena |
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Institution: | 1 Dipartimento di Chimica, Università degli Studi di Napoli “Federico II,” Via Cintia, 80126 Naples, Italy 2 Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli “Federico II,” Via Cintia, 80126 Naples, Italy 3 Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 6, 80134 Naples, Italy |
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Abstract: | Bovine seminal ribonuclease exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein. Surprisingly, the cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer. This buried diversity comes into light in the reducing environment of the cytosol, where the unswapped dimer dissociates into monomers, whereas the swapped one generates a metastable dimeric form (NCD-BS) with a quaternary assembly that allows the molecule to escape the protein inhibitor of ribonucleases. The stability of this quaternary shape was mainly attributed to the combined presence of Pro19 and Leu28. We have prepared and fully characterized by X-ray diffraction the double mutant P19A/L28Q (PALQ) of the seminal enzyme. While the swapped and unswapped forms of the mutant have structures very similar to that of the corresponding wild-type forms, the non-covalent form (NCD-PALQ) adopts an opened quaternary structure, different from that of NCD-BS. Moreover, model building clearly indicates that NCD-PALQ can be easily sequestered by the protein inhibitor. In agreement with these results, cytotoxic assays have revealed that PALQ has limited activity, whereas the single mutants P19A and L28Q display cytotoxic activity against malignant cells almost as large as the wild-type enzyme. The significant increase in the antitumor activity, brought about by the substitution of just two residues in going from the double mutant to the wild-type enzyme, suggests a new strategy to improve this important biological property by strengthening the interface that stabilizes the quaternary structure of NCD-BS. |
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Keywords: | 3-D three-dimensional BS-RNase bovine seminal ribonuclease CD-RNaseA C-terminal swapped dimer of bovine pancreatic ribonuclease M× M-BS dimeric form of BS-RNase in which the chains swap their N-terminal tails M=M-BS unswapped dimer of BS-RNase M× M-PALQ dimeric form of mutant P19A/L28Q of BS-RNase in which the chains swap their N-terminal tails M=M-PALQ unswapped dimer of P19A/L28Q of BS-RNase NCD-BS non-covalent swapped form of BS-RNase NCD-L28Q non-covalent swapped form of the L28Q mutant of BS-RNase NCD-PALQ non-covalent swapped form of the P19A/L28Q mutant of BS-RNase NCD-P19A non-covalent swapped form of the P19A mutant of BS-RNase ND-RNaseA N-terminal swapped dimer of bovine pancreatic ribonuclease RI ribonuclease inhibitor RNase A bovine pancreatic ribonuclease PDB Protein Data Bank PM8 N-terminal swapped dimer of a variant of human pancreatic ribonuclease MTT (3-(4 5-Dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide) |
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