Structure–cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants

Abstract

Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribo-nucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity. Abbreviations BS-RNase, bovine seminal ribonuclease; DSC, differential scanning calorimetry; GSH, glutathione; hA-BS-RNase, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; hA-mBS, G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; mBS, monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; MxM, dimeric form of bovine seminal ribonuclease in which the chains swap their N-termini; M=M, unswapped dimer of bovine seminal ribonuclease; NCD, noncovalent dimer; PDB, Protein Data Bank; RI, ribonuclease inhibitor; RNase A, bovine pancreatic ribonuclease; S 80-BS-RNase, R80S variant of bovine seminal ribonuclease; S 80-hA-BS-RNase, R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of bovine seminal ribonuclease; S 80-hA-mBS, R80S ⁄ G16S ⁄ N17T ⁄ P19A ⁄ S20A variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties; S 80-mBS, R80S variant of the monomeric N67D variant of bovine seminal ribonuclease with Cys31 and Cys32 linked to glutathione moieties.

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

“…In addition, the monitoring of urea denaturation of mBS by NMR allowed us to follow the unfolding process in a residue specific way and to elucidate, eventually, the presence of residues more prone to unfolding and possibly prompting dislocation and swapping. The analysis reported in figure 4 gives us a convincing evidence of a two-step denaturation mechanism for all the protein regions, in agreement with a recent CD and calorimetric study [28], showing that the protein denaturation follows a two step mechanism, and excluding the presence of significantly populated intermediates. On the whole, all data presented in this paper definitely confirm a close similarity with RNase A structure, suggesting also that this protein is extremely compact and there is no evidence of a pre-opening of the monomeric structure in solution.…”

“…With respect to our previous data [21], this more detailed investigation highlights the presence of potential hot-spots along the protein structure, in particular in the C-terminal region, henceforth we cannot exclude the possibility that, in different and eventually more severe conditions, also the C-terminal swapping could be observed. Regarding the N-terminal hinge region, this is still one of the most flexible regions of the protein but, according to previous mutagenesis studies [19], [48], hinge flexibility together with swapping propensity and in vitro antitumor activity [28], are not strictly dependent on a specific sequence.…”

“…Inspired by these observations, we have engineered the BS-RNase sequence by substituting either all the different residues of the hinge loop or only the central Pro at position 19, with the corresponding residues of RNase A. Surprisingly, none of these variants displayed significant differences in the hinge region flexibility and swapping extent [19], [27], and the biological activity was only marginally affected [28]. A similar behaviour has been observed also in other swapped proteins, leading to general conclusion that “…based on mutagenesis studies, it is doubtful that sequence features alone determine whether a protein will undergo domain swapping” [2].…”

Structural and functional relationships of natural and artificial dimeric bovine ribonucleases: New scaffolds for potential antitumor drugs

“…In addition, the BS‐RNase and RNase A variants with Arg80 display a slightly increased percentage of swapped form compared to the corresponding Ser80 variants [23,59,86], and also the dissociation rate of the corresponding NCD forms is decreased [23,59,87]. Overall, the data confirm that, at least for RNase A, 3D‐DS is an essential requisite for cytotoxicity, because it stabilizes the dimeric structure even in the reducing environment of the cytosol.…”

Structural determinants of the eosinophil cationic protein antimicrobial activity

“…The proteins would act as a reservoir of active peptides (Pizzo and D ' Alessio, 2007 ;Pizzo et al , 2008 ;Rosenberg , 2008b ;Giancola et al , 2011 ;Sanchez et al , 2011 ). This proposal relies on the observation that some antimicrobial proteins retain their activity or are only active when denatured (During et al , 1999 ;Schroeder et al , 2011 ).…”