Onconase: An Unusually Stable Protein ² Eugenio Notomista, ‡ Francesca Catanzano, § Giuseppe Graziano, | Fabrizio Dal Piaz, ‡,⊥ Guido Barone, § Giuseppe D’Alessio, ‡ and Alberto Di Donato* ,‡ Dipartimento di Chimica Organica e Biologica, UniVersita ` di Napoli Federico II, Via Mezzocannone 16, 80134 Naples, Italy, Dipartimento di Chimica, UniVersita ` di Napoli Federico II, Via Mezzocannone, 4-80134 Naples, Italy, Facolta ` di Scienze, UniVersita ` del Sannio Via Port’Arsa, 11-82100 BeneVento, Italy, and Centro Internazionale di SerVizi di Spettrometria di Massa, Via Pansini, 5. 80131 Naples, Italy ReceiVed February 23, 2000; ReVised Manuscript ReceiVed May 3, 2000 ABSTRACT: Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity. At least four members of the RNase A (bovine pancreatic ribonuclease) 1 superfamily are endowed with antitumor activity and show selective cytotoxicity toward several tumor cell lines: bovine seminal ribonuclease (BS-RNase) from bull semen, onconase (ONC) from oocytes of Rana pipiens, and the closely related sialic acid-binding lectins from oocytes of Rana catesbeiana and Rana japonica (1). These proteins have been extensively studied because of their potential as antitumor drugs, and ONC has reached phase- III clinical trials (2). ONC is the smallest member of the RNase A superfamily, with 104 residues as compared to the 124 in the primary structure of RNase A, and shares 30% identity with the RNase A sequence (3). Despite the low degree of identity between their primary structures, the three-dimensional structure of ONC (4) shows a topology very similar to that of RNase A (Figure 1), with the major differences present in the loop regions and at the C-terminus, where ONC has an additional disulfide bond (Cys87-Cys104), which is found only in frog RNases (4, 5). It should be noted that a strong similarity in three-dimensional structures is a common trait in the superfamily. Thus, the RNases show divergent primary structures (6, 7) as well as a well-preserved tertiary fold marked by a core structure made up of two antiparallel -sheets and three R-helices. It has been reported (8) that ONC has an unusually high denaturation temperature, which positions ONC among the most thermostable mesophilic proteins found so far. Indeed, if the high T d measured for ONC is a consequence of its stability, this might well be the cause for both the low catalytic activity of the protein (9, 10) and its renal toxicity (11). In fact, if the stability of the protein is based on its intrinsic rigidity, as is the case for most thermophilic proteins (12), this could explain both (i) its low activity, and thus, its low flexibility in adapting to the substrate and/or to the transition state, the key event in the catalytic mechanism ( 13), and (ii) its renal toxicity, for the difficulty in clearing the protein, resulting in its high concentration in the kidney, which can lead to renal damage. To investigate the determinants of the stability of ONC, we studied by differential scanning calorimetry, circular dichroism, and fluorescence measurements the main ther- modynamic parameters of the protein and of a mutant, ² This work was supported by grants from the Ministry of University and Research (PRIN/97, SMIP, and PRIN/97, CFSIB), the MURST- CNR Program L. 95/95, and the National Research Council (PF- Biotecnologie). * To whom correspondence should be addressed. Phone: +39-081- 7041 271. Fax: +39-081-552 1217. E-mail: didonato@unina.it. ‡ Dipartimento di Chimica Organica e Biologica, Universita ` di Napoli Federico II. § Dipartimento di Chimica, Universita ` di Napoli Federico II. | Facolta ` di Scienze, Universita ` del Sannio. ⊥ Centro Internazionale di Servizi di Spettrometria di Massa. 1 BS-RNase, bovine seminal ribonuclease; RNase A, bovine pan- creatic ribonuclease A; ONC, Onconase; (M23L)-ONC, mutant of onconase with leucine replacing methionine at position 23; (Q1E)-ONC, mutant of onconase with glutamic acid replacing glutamine at position 1; ES/MS, electrospray mass spectrometry; DSC, differential scanning calorimetry; CD, circular dichroism; ASA, accessible surface area; MES, 2-(N-morpholino)ethanesulfonic acid; HEPPSO, N-(2-hydroxy- ethyl)piperazine-N′-(2-hydroxypropanesulfonic acid); Tris, tris(hy- droxymethyl)aminomethane; PMSF, phenylmethanesulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; CNBr, cyanogen bromide; DTT, dithiothreitol; TFA, trifluoroacetic acid; GuHCl, guanidinium chloride; RP-HPLC, reverse-phase high-performance liquid chroma- tography; PDB, Protein Data Bank. 8711 Biochemistry 2000, 39, 8711-8718 10.1021/bi000415x CCC: $19.00 © 2000 American Chemical Society Published on Web 07/06/2000