The X-ray structure of the non-covalent dimeric form of S16G,A19P,Q28L,R31C,S32C mutant of RNase A re-opens the question of structure-cytotoxic activity relationship in dimeric RNases

Pancreatic ribonucleases, thanks to their ability to degrade RNA, represent potential antitumor therapeutics. It has been suggested that RNases have to reach the cytosol and to avoid the binding of the ribonuclease inhibitor (RI) in order to exert their cytotoxic activity1. Among antitumor RNases, bovine seminal ribonuclease (BS-RNase) is an interesting case: it is a covalent homodimer with two interchain antiparallel disulphide bridges between Cys31 and Cys322-3, which in the reducing cellular compartment of cytosol convert into a non covalent dimeric form (NCD) stabilized by the swapping of N-terminal helices4. It has been suggested that NCD-BS avoids inhibition by RI because it adopts a compact quaternary structure that interferes with binding [4]. Structural studies have indicated a key role in determining this dimeric assembly for specific amino acid residues: Pro19 and Leu285. Using the homology mutation approach, few structural features of BS-RNase have been introduced in bovine pancreatic ribonuclease (RNase A), which does not show antitumor action. The introduction of Leu28, Cys31 and Cys32 (LCC) and that of the same residues plus Pro19 (PLCC) or the introduction of the hinge peptide sequence (residues 16-22) of BS-RNase and the two cysteines (GNPSCC) in the sequence of RNase A produced dimeric variants which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase6-7. Structural determination of NCD forms of these mutants has allowed to rationalyze their biological activities: all the dimers have open quaternary structures, which can be easily sequestered by RI. The comparison of these structures with those previously reported for other RNase swapped dimers has suggested that an additional important role in determining the compact quaternary structure of NCD-BS can be played also by Gly167. Therefore, the GPLCC mutant of RNase A (with substitution S16G, A19P, Q28L, R31C and S32C) has been produced and found to exert a cytotoxic activity on SVT2 cells comparable to that of seminal enzyme. In order to verify whether NCDGPLCC has a quaternary assembly similar to NCD-BS, its structure has been determined from two different crystal forms obtained from similar crystallization conditions. Surprisingly, NCD-GPLCC shows two different quaternary structures, both of them distinct with respect to NCD-BS. As predicted7, the introduction of Gly16 determines a hinge peptide conformation similar to that observed in NCD-BS. However, in each structure this occurs only for one of the two hinge peptides, and the resulting quaternary assembly is still compatible with the RI binding. Altogether these data represent an encouraging step toward a rational design of a dimeric form of RNase A endowed with a strong cytotoxic activity and point up the necessity of a deeper characterization of its complex with RI, in terms of binding constants and residual ribonuclease activity.