The buried diversity of bovine seminal ribonuclease: Shape and cytotoxicity of the swapped non-covalent form of the enzyme(451 views) Merlino A, Ercole C, Picone D, Pizzo E, Mazzarella L, Sica F
Keywords: 3-D Domain Swapping, Antitumor Activity, Bovine Seminal Ribonuclease, Crystal Structure, Non-Covalent Dimer, Enzyme, Leucine, Proline, Protein L 28 Q, Protein Palq, Unclassified Drug, Animal Cell, Antineoplastic Activity, Article, Cancer Cell, Controlled Study, Cytosol, Cytotoxicity, Mouse, Nonhuman, Priority Journal, Seminal Vesicle, Wild Type, X Ray Diffraction, 3t3 Cells, Antigens, Polyomavirus Transforming, Buffers, Cattle, Cell Line, Cell Survival, Cell Transformation, Dimerization, Disulfides, Endoribonucleases, Enzyme Stability, Formazans, Heat, Hydrogen Bonding, Hydrogen-Ion Concentration, Isomerism, Kinetics, Inbred Balb C, Models, Chemical, Molecular, Mutation, Protein Conformation, Protein Folding, Protein Structure, Quaternary, Secondary, Tertiary, Recombinant Proteins, Temperature, Tetrazolium Salts, Time Factors, Tromethamine, Variation (genetics), Water, X-Ray Diffraction,
Affiliations: *** IBB - CNR ***
Dipartimento di Chimica, Università degli Studi di Napoli Federico II, Via Cintia, 80126 Naples, Italy
Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli Federico II, Via Cintia, 80126 Naples, Italy
Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 6, 80134 Naples, Italy
References: Arnold, U., Ulrich-Hofmann, R., Natural and engineered ribonucleases as potential cancer therapeutics (2006) Biotechnol. Lett., 28, pp. 1615-162
Makarov, A.A., Ilinskaya, O.N., Cytotoxic ribonucleases: molecular weapons and their targets (2003) FEBS Lett., 540, pp. 15-20
Leland, P.A., Raines, R.T., Cancer chemotherapy-ribonucleases to the rescue (2001) Chem. Biol., 8, pp. 405-413
Bretscher, L.E., Abel, R.L., Raines, R.T., A ribonuclease A variant with low catalytic activity but high cytotoxicity (2000) J. Biol. Chem., 275, pp. 9893-9896
Merlino, A., Mazzarella, L., Carannante, A., Di Fiore, A., Di Donato, A., Notomista, E., Sica, F., The importance of dynamic effects on the enzyme activity: X-ray structure and molecular dynamics of onconase mutants (2005) J. Biol. Chem., 280, pp. 17953-17960
Leland, P.A., Schultz, L.W., Kim, B.M., Raines, R.T., Ribonuclease A variants with potent cytotoxic activity (1998) Proc. Natl Acad. Sci. USA, 95, pp. 10407-10412
Bosch, M., Benito, A., Ribo, M., Puig, T., Beaumelle, B., Vilanova, M., A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity (2004) Biochemistry, 43, pp. 2167-2177
Rodriguez, M., Benito, A., Tubert, P., Castro, J., Ribo, M., Beaumelle, B., Vilanova, M., A cytotoxic ribonuclease variant with a discontinuous nuclear localization signal constituted by basic residues scattered over three areas of the molecule (2006) J. Mol. Biol., 360, pp. 548-557
Newton, D.L., Xue, Y., Boqué, L., Wlodawer, A., Kung, H.F., Rybak, S.M., Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy (1997) Protein Eng., 10, pp. 463-470
Gaur, D., Swaminathan, S., Batra, J.K., Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants (2001) J. Biol. Chem., 276, pp. 24978-24984
Matousek, J., Gotte, G., Pouckova, P., Soucek, J., Slavick, T., Vottariello, F., Libonati, M., Antitumor and other biological actions of oligomers of RNase A (2003) J. Biol. Chem., 278, pp. 23817-23822
Libonati, M., Biological actions of the oligomers of ribonuclease A (2004) Cell. Mol. Life Sci., 61, p. 2431
Libonati, M., Gotte, G., Oligomerization of bovine ribonuclease A: structural and functional features of its multimers (2004) Biochem. J., 380, pp. 311-327
Piccoli, R., Di Gaetano, S., De Lorenzo, C., Grauso, M., Monaco, C., Spalletti-Cernia, D., A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells (1999) Proc. Natl Acad. Sci. USA, 96, pp. 7768-7773
Piccoli, R., Vescia, S., Bridges, S.H., D'Alessio, G., The antitumor action of seminal ribonuclease tested with the plasmacytoma spleen colonization assay (1990) Ital. J. Biochem., 39, pp. 242-249
Cafaro, V., De Lorenzo, C., Piccoli, R., Bracale, A., Mastronicola, M.R., Di Donato, A., D'Alessio, G., The antitumor action of seminal ribonuclease and its quaternary conformations (1995) FEBS Lett., 359, pp. 31-34
Mazzarella, L., Capasso, S., Demasi, D., Di Lorenzo, G., Mattia, C.A., Zagari, A., Bovine seminal ribonuclease: structure at 1.9 Å resolution (1993) Acta Crystallogr., Sect. D: Biol. Crystallogr., 49, pp. 389-402
Berisio, R., Sica, F., De Lorenzo, C., Di Fiore, A., Piccoli, R., Zagari, A., Mazzarella, L., Crystal structure of the dimeric unswapped form of bovine seminal ribonuclease (2003) FEBS Lett., 554, pp. 105-110
Piccoli, R., Tamburrini, M., Piccialli, G., Di Donato, A., Parente, A., D'Alessio, G., The dual-mode quaternary structure of seminal RNase (1992) Proc. Natl Acad. Sci. USA, 89, pp. 1870-1874
Merlino, A., Vitagliano, L., Sica, F., Zagari, A., Mazzarella, L., Population shift versus induced fit: the case of bovine seminal ribonuclease swapping dimer (2004) Biopolymers, 73, pp. 689-695
Sica, F., Di Fiore, A., Merlino, A., Mazzarella, L., Structure and stability of the non-covalent swapped dimer of bovine seminal ribonuclease: an enzyme tailored to evade ribonuclease protein inhibitor (2004) J. Biol. Chem., 279, pp. 36753-36760
Liu, Y., Gotte, G., Libonati, M., Eisenberg, D., A domain-swapped RNase A dimer with implications for amyloid formation (2001) Nat. Struct. Biol., 8, pp. 211-214
Liu, Y., Hart, P.J., Schlunegger, M.P., Eisenberg, D., The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1 Å resolution (1998) Proc. Natl Acad. Sci. USA, 95, pp. 3437-3442
Canals, A., Pous, J., Guasch, A., Benito, A., Ribo, M., Vilanova, M., Coll, M., The structure of an engineered domain-swapped ribonuclease dimer and its implications for the evolution of proteins toward oligomerization (2001) Structure, 9, pp. 967-976
Naddeo, M., Vitagliano, L., Russo, A., Gotte, G., D'Alessio, G., Sorrentino, S., Interactions of the cytotoxic RNase A dimers with the cytosolic ribonuclease inhibitor (2005) FEBS Lett., 579, pp. 2663-2668
Ercole, C., Avitabile, F., Del Vecchio, P., Crescenzi, O., Tancredi, T., Picone, D., Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase (2003) Eur. J. Biochem., 270, pp. 4729-4735
Picone, D., Di Fiore, A., Ercole, C., Franzese, M., Sica, F., Tomaselli, S., Mazzarella, L., The role of the hinge loop in domain swapping. The special case of bovine seminal ribonuclease (2005) J. Biol. Chem., 280, pp. 13771-13778
Bennett, M.J., Schlunegger, M.P., Eisenberg, D., 3D Domain swapping: a mechanism for oligomer assembly (1995) Protein Sci., 4, pp. 2455-2468
Liu, Y., Eisenberg, D., 3D domain swapping: as domains continue to swap (2002) Protein Sci., 11, pp. 1285-1299
Avitabile, F., Alfano, C., Spadaccini, R., Crescenzi, O., D'Ursi, A.M., D'Alessio, G., The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases (2003) Biochemistry, 42, pp. 8704-8711
Otwinowski, Z., Minor, W., Processing of x-ray diffraction data collected in oscillation mode (1997) Methods Enzymol., 276, pp. 307-326
Navaza, J., Saludjian, P., AMoRe: an automated molecular replacement program package (1997) Methods Enzymol., 276, pp. 581-594
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Crystallography & NMR system: a new software suite for macromolecular structure determination (1998) Acta Crystallogr., Sect. D: Biol. Crystallogr. D, 54, pp. 905-921
Jones, T.A., Bergdoll, M., Kjeldgaard, M., O: a macromolecular modeling environment (1990) Crystallographic and Modeling Methods in Molecular Design, pp. 189-199. , Bugg C., and Ealick S. (Eds), Springer-Verlag Press, Heidelberg
Laskowski, R.A., MacArthur, M.W., Moss, M.D., Thorton, J.M., PROCHECK: a program to check the stereochemical quality of protein structure (1993) J. Appl. Crystallogr., 26, pp. 283-291
Hooft, R.W., Vriend, G., Sander, C., Abola, E.E., Errors in protein structures (1996) Nature, 381, p. 272
Kobe, B., Deisenhofer, J., Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A (1996) J. Mol. Biol., 264, pp. 1028-1043
Kraulis, P.J., MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures (1991) J. Appl. Crystallogr., 24, pp. 946-950
Esnouf, R.M., Further additions to MolScript version 1.4, including reading and contouring of electron-density maps (1999) Acta Crystallogr., Sect. D: Biol. Crystallogr., 55, pp. 938-940
McDonald, I.K., Thornton, J.M., Satisfying hydrogen bonding potential in proteins (1994) J. Mol. Biol., 238, pp. 777-793
Brunger, A.T., (1996) X-PLOR Version 3.8, , Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT
Adinolfi, B.S., Cafaro, V., D'Alessio, G., Di Donato, A., Full antitumor action of recombinant seminal ribonuclease depends on the removal of its N-terminal methionine (1995) Biochem. Biophys. Res. Commun., 213, pp. 525-532
Abe, K., Kimura, H., Amyloid beta toxicity consists of a Ca(2+)-independent early phase and a Ca(2+)-dependent late phase (1996) J. Neurochem., 67, pp. 2074-2078
Bracale, A., Spalletti-Cernia, D., Mastronicola, M., Castaldi, F., Mannucci, R., Nitsch, L., D'Alessio, G., Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease (2002) Biochem. J., 362, pp. 553-560
Makarov, A. A., Ilinskaya, O. N., Cytotoxic ribonucleases: molecular weapons and their targets (2003) FEBS Lett., 540, pp. 15-20
Leland, P. A., Raines, R. T., Cancer chemotherapy-ribonucleases to the rescue (2001) Chem. Biol., 8, pp. 405-413
Bretscher, L. E., Abel, R. L., Raines, R. T., A ribonuclease A variant with low catalytic activity but high cytotoxicity (2000) J. Biol. Chem., 275, pp. 9893-9896
Leland, P. A., Schultz, L. W., Kim, B. M., Raines, R. T., Ribonuclease A variants with potent cytotoxic activity (1998) Proc. Natl Acad. Sci. USA, 95, pp. 10407-10412
Newton, D. L., Xue, Y., Boqu, L., Wlodawer, A., Kung, H. F., Rybak, S. M., Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy (1997) Protein Eng., 10, pp. 463-470
Liu, Y., Hart, P. J., Schlunegger, M. P., Eisenberg, D., The crystal structure of a 3D domain-swapped dimer of RNase A at a 2. 1 resolution (1998) Proc. Natl Acad. Sci. USA, 95, pp. 3437-3442
Bennett, M. J., Schlunegger, M. P., Eisenberg, D., 3D Domain swapping: a mechanism for oligomer assembly (1995) Protein Sci., 4, pp. 2455-2468
McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., Read, R. J., Likelihood-enhanced fast translation functions (2005) Acta Crystallogr., Sect. D: Biol. Crystallogr. D, 61, pp. 458-464
Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Crystallography & NMR system: a new software suite for macromolecular structure determination (1998) Acta Crystallogr., Sect. D: Biol. Crystallogr. D, 54, pp. 905-921
Jones, T. A., Bergdoll, M., Kjeldgaard, M., O: a macromolecular modeling environment (1990) Crystallographic and Modeling Methods in Molecular Design, pp. 189-199. , Bugg C., and Ealick S. (Eds), Springer-Verlag Press, Heidelberg
Laskowski, R. A., MacArthur, M. W., Moss, M. D., Thorton, J. M., PROCHECK: a program to check the stereochemical quality of protein structure (1993) J. Appl. Crystallogr., 26, pp. 283-291
Hooft, R. W., Vriend, G., Sander, C., Abola, E. E., Errors in protein structures (1996) Nature, 381, p. 272
Kraulis, P. J., MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures (1991) J. Appl. Crystallogr., 24, pp. 946-950
Esnouf, R. M., Further additions to MolScript version 1. 4, including reading and contouring of electron-density maps (1999) Acta Crystallogr., Sect. D: Biol. Crystallogr., 55, pp. 938-940
McDonald, I. K., Thornton, J. M., Satisfying hydrogen bonding potential in proteins (1994) J. Mol. Biol., 238, pp. 777-793
Brunger, A. T., (1996) X-PLOR Version 3. 8, , Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT
Adinolfi, B. S., Cafaro, V., D'Alessio, G., Di Donato, A., Full antitumor action of recombinant seminal ribonuclease depends on the removal of its N-terminal methionine (1995) Biochem. Biophys. Res. Commun., 213, pp. 525-532
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. (C) 2007 Elsevier Ltd. All rights reserved.
The buried diversity of bovine seminal ribonuclease: Shape and cytotoxicity of the swapped non-covalent form of the enzyme