The buried diversity of bovine seminal ribonuclease: shape and cytotoxicity of the swapped non-covalent form of the enzyme.
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.
Merlino
Antonello
A
Dipartimento di Chimica, Università degli Studi di Napoli Federico II, Via Cintia, 80126 Naples, Italy.
Ercole
Carmine
C
Picone
Delia
D
Pizzo
Elio
E
Mazzarella
Lelio
L
Sica
Filomena
F
D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology; D002021:Buffers; D002417:Cattle; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D002472:Cell Transformation, Viral; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity*; D004795:Enzyme Stability; D005562:Formazans / Q000378:metabolism; D014644:Genetic Variation; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D013696:Temperature; D013778:Tetrazolium Salts / Q000378:metabolism; D013997:Time Factors; D014325:Tromethamine / Q000737:chemistry; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction
D000952:Antigens, Polyomavirus Transforming; D002021:Buffers; D004220:Disulfides; D005562:Formazans; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D014325:Tromethamine; D014867:Water; C079782:MTT formazan; D004722:Endoribonucleases; C032523:ribonuclease SPL