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

BS-RNase, a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, MxM and M=M, with and without exchange of the monomers’ N-termini. In the cytosolic reducing environment, MxM is converted into a non-covalent 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 (RI), a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction M=M is converted in two monomers, which are readily bound and inactivated by RI. Based on 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 substitution of Arg80 with Ser decreases significantly 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 nor 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.