Leucine 145 of the ribotoxin α-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Protein Science

Volumn 12, Issue 1, 2003, Pages 161-169

  Masip, Manuel ^a   García Ortega, Lucía ^a   Olmo, Nieves ^a   García Mayoral, Ma Flor ^b   Pérez Cañadillas, José Manuel ^b   Bruix, Marta ^b   Oñaderra, Mercedes ^a   Martínez del Pozo, Álvaro ^a   Gavilanes, José G ^a  

^a UNIVERSIDAD COMPLUTENSE DE MADRID   (Spain)

^b INSTITUTO DE QUÍMICA FÍSICA ROCASOLANO   (Spain)

Author keywords

Cytotoxic protein; Ribonuclease; Ribotoxins; RNase T1; RNase U2

Indexed keywords

ALPHA SARCIN; CYTOTOXIC FACTOR; LEUCINE; MUTANT PROTEIN; RIBONUCLEASE; RIBOSOME INACTIVATING PROTEIN; RIBOTOXIN; UNCLASSIFIED DRUG;

ARTICLE; CIRCULAR DICHROISM; ENZYME ACTIVE SITE; FLUORESCENCE SPECTROSCOPY; NONHUMAN; NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN CONFORMATION; SPECTROSCOPY;

AMINO ACID SUBSTITUTION; BINDING SITES; CIRCULAR DICHROISM; ENDORIBONUCLEASES; FUNGAL PROTEINS; FUNGI; HEAT; HUMANS; HYDROGEN-ION CONCENTRATION; INHIBITORY CONCENTRATION 50; LEUCINE; MODELS, MOLECULAR; PROTEIN CONFORMATION; PROTEIN DENATURATION; RHABDOMYOSARCOMA; RIBOSOMES; RNA STABILITY; RNA, RIBOSOMAL; TUMOR CELLS, CULTURED;

EID: 0037214144     PISSN: 09618368     EISSN: None     Source Type: Journal    
DOI: 10.1110/ps.0225903     Document Type: Article

References (41)

1. Bax, A. and Davies, D.G. 1985. MLEV-17 based two-dimensional homonuclear magnetisation transfer spectroscopy. J. Magn. Reson. 65: 355-360.
2. Becktel, W.J. and Schellman, J.A. 1987. Protein stability curves. Biopolymers 26: 1859-1877.
3. 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease α-sarcin. Protein Sci. 5: 969-972.
4. Correll, C.C., Munishkin, A., Chan, Y.L., Ren, Z., Wool, I.G., and Steitz, T.A. 1998. Crystal structure of the ribosomal RNA domain essential for binding elongation factors. Proc. Natl. Acad. Sci. 95:13436-13441.
5. Correll, C.C., Wool, I.G., and Munishkin, A. 1999. The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: The structure at 1.11 Δ resolution. J. Mol. Biol. 292: 275-287.
6. Doumen, J., Gonciarz, M., Zegers, I., Loris, R., Wyns, L., and Steyaert, J. 1996. A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1. Protein Sci. 5: 1523-1530.
7. Endo, Y., Hubert, P.W., and Wool, I.G. 1983. The ribonuclease activity of the cytotoxin α-sarcin: The characteristics of the enzymatic activity of α-sarcin with ribosomes and ribonucleic acids as substrates. J. Biol. Chem. 258: 2662-2667.
8. García-Ortega, L., Lacadena, J., Lacadena, V., Masip, M., de Antonio, C., Martínez-Ruiz, A., and Martínez del Pozo, A. 2000. The solubility of the ribotoxin α-sarcin, produced as a recombinant protein in Escherichia coli, is significantly increased in the presence of thioredoxin. Lett. Appl. Microbiol. 30: 298-302.
9. García-Ortega, L., Lacadena, J., Mancheño, J.M., Oñaderra, M., Kao, R., Davies, J., Olmo, N., Martínez del Pozo, A., and Gavilanes, J.G. 2001. Involvement of the amino-terminal β-hairpin of the Aspergillus ribotoxins on the interaction with membranes and non-specific ribonuclease activity. Protein Sci. 10: 1658-1668.
10. 2-terminal β-hairpin of the ribotoxin α-sarcin produces a non-toxic but active ribonuclease. J. Biol. Chem. 277: 18632-18639.
11. Gasset, M., Mancheño, J.M., Lacadena, J., Turnay, J., Olmo, N., Lizarbe, M.A., Martínez del Pozo, A., Oñaderra, M., and Gavilanes, J.G. 1994. α-sarcin, a ribosome-inactivating protein that translocates across the membrane of phospholipid vesicles. Current Topics in Peptide and Protein Research 1: 99-104.
12. Inagaki, F., Kawano, Y., Shimada, I., Takahashi, K., and Miyazawa, T. 1981. Nuclear magnetic resonance study on the microenvironments of histidine residues of ribonuclease T1 and carboxymethylated ribonuclease T1. J. Biochem. (Tokyo) 89: 1185-1195.
13. Kao, R. and Davies, J. 1995. Fungal ribotoxins: A family of naturally engineered toxins? Biochem. Cell Biol. 73: 1151-1159.
14. -, 1999. Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonucleases. J. Biol. Chem. 274: 12576-12582.
15. -, 2000. Single amino acid substitution affecting the specificity of the fungal ribotoxin mitogillin. FEBS Lett. 466: 87-90.
16. Kao, Y.-H., Fitch, C.A., Bhattacharya, S., Sarkisian, C.J., Lecomte, J.T.J., and García-Moreno, E.B. 2000. Salt effects on ionization equilibria of histidines in myoglobin. Biophys. J. 79: 1637-1654.
17. Kao, R., Martínez-Ruiz, A., Martínez del Pozo, A., Crameri, R., and Davies, J. 2001. Mitogillin and related fungal ribotoxins. Methods Enzymol. 341: 324-335.
18. Koradi, R., Billeter, M., and Wütrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graph. 14: 51-55.
19. Kumar, A., Ernst, R.R., and Wüthrich, K. 1980. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross relaxation networks in biological macromolecules. Biochem. Biophys. Res. Commun. 95: 1-6.
20. Kumar, K. and Walz, Jr., F.G. 2001. Probing functional perfection in substructures of ribonuclease T1: Double combinatorial random mutagenesis involving Asn43, Asn44, and Glu46 in the guanine binding loop. Biochemistry 40: 3748-3757.
21. Lacadena, J., Martínez del Pozo, A., Barbero, J.L., Mancheño, J.M., Gasset, M., Oñaderra, M., López-Otín, C., Ortega, S., García, J.L., and Gavilanes, J.G. 1994. Overproduction and purification of biologically active native fungal α-sarcin in Escherichia coli. Gene 142: 147-151.
22. Lacadena, J., Martínez del Pozo, A., Lacadena, V., Martínez-Ruiz, A., Mancheño, J.M., Oñaderra, M., and Gavilanes, J.G. 1998. The cytotoxin α-sarcin behaves as a cyclizing ribonuclease. FEBS Lett. 424: 46-48.
23. Lacadena, J., Martínez del Pozo, A., Martínez-Ruiz, A., Pérez-Cañadillas, J.M., Bruix, M., Mancheño, J.M., Oñaderra, M., and Gavilanes, J.G. 1999. Role of histidine-50, glutamic acid-96 and histidine-137 in the ribonucleolytic mechanism of the ribotoxin α-sarcin. Proteins 37: 474-484.
24. Lamy, B., Davies, J., and Schindler, D. 1992. The Aspergillus ribonucleolytic toxins (ribotoxins). In Genetically Engineered Toxins (ed. A.E. Frankel), pp. 237-257. Marcel Dekker, New York, NY.
25. Lee, K.K., Fitch, C.A., Lecomte, J.T.J., and García-Moreno, E.B. 2002. Electrostatic effects in highly charged proteins: Salt sensitivity of pKa values of histidines in staphylococcal nuclease. Biochemistry 41: 5656-5657.
26. Martínez del Pozo, A., Gasset, M., Oñaderra, M., and Gavilanes, J.G. 1988. Conformational study of the antitumor protein α-sarcin. Biochim. Biophys. Acta 953: 280-288.
27. Martínez-Ruiz, A., Kao, R., Davies, J., and Martínez del Pozo, A. 1999. Ribotoxins are a more widespread group of proteins within the filamentous fungi than previously believed. Toxicon 37: 1549-1563.
28. Martínez-Ruiz, A., García-Ortega, L., Kao, R., Lacadena, J., Oñaderra, M., Mancheño, J.M., Davies, J., Martínez del Pozo, A., and Gavilanes, J.G. 2001. RNase U2 and α-sarcin: A study of relationships. Methods Enzymol. 341: 335-351.
29. Masip, M., Lacadeña, J., Mancheño, J.M., Oñaderra, M., Martínez-Ruiz, A., Martínez del Pozo, A, and Gavilanes. J.G. 2001. Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin α-sarcin. Eur. J. Biochem. 268: 1-8.
30. Olmo, N., Turnay, J., González de Buitrago, G., López de Silanes, I., Gavilanes, J.G., and Lizarbe, M.A. 2001. Cytotoxic mechanism of the ribotoxin α-sarcin. Induction of cell death via apoptosis. Eur. J. Biochem. 268: 2113-2123.
31. Olson, B.H. and Goemer, G.L. 1965. α-sarcin, a new antitumor agent. I. Isolation, purification, chemical composition, and the identity of a new amino acid. Appl. Microbiol. 13: 314-321.
32. a values and titration shifts in the cytotoxic ribonuclease α-sarcin by NMR. Relationship between electrostatic interactions, structure and catalytic function. Biochemistry 37: 15865-15876.
33. Pérez-Cañadillas, J.M., Santoro, J., Campos-Olivas, R., Lacadena, J., Martínez del Pozo, A., Gavilanes, J.G., Rico, M., and Bruix, M. 2000. The highly refined solution structure of the cytotoxic ribonuclease α-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity. J. Mol. Biol. 299: 1061-1073.
34. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
35. Schindler, D.G. and Davies, J.E. 1977. Specific cleavage of ribosomal RNA caused by α-sarcin. Nucleic Acids Res. 4: 1097-1100.
36. Steyaert, J. 1997. A decade of protein engineering on ribonuclease T1. Atomic dissection of the enzyme-substrate interaction. Eur. J. Biochem. 247: 1-11.
37. Turnay, J., Olmo, N., Jiménez, A., Lizarbe, M.A., and Gavilanes, J.G. 1993. Kinetic study of the cytotoxic effect of α-sarcin, a ribosome inactivating protein from Aspergillus giganteus, on tumour cell lines: Protein biosynthesis inhibition and cell binding. Mol. Cell. Biochem. 122: 39-47.
38. Wüthrich, K. 1986. NMR of proteins and nucleic acids. Wiley, New York.
39. Yang, X.J. and Moffat, K. 1996. Insights into specificity of cleavage and mechanism of cell entry from the crystal structure of the highly specific Aspergillus ribotoxin, restrictocin. Structure 4: 837-852.
40. Yang, X., Gérczei, T., Glover, L.T., and Correll, C.C. 2001. Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping. Nat. Struct. Biol. 8: 968-973.
41. Yoshida, H. 2001. The ribonuclease T1 family. Methods Enzymol. 341: 28-41.