Structural aspects of covalent and non-covalent dimers of pancreaticribonucleases

The ability to retain ribonucleolytic activity in the presence of the ribonuclease inhibitor (RI) is one of the determinant of ribonucleases (RNases) cytotoxicity. Bovine seminal ribonuclease (BSRNase), the only dimeric protein among the pancreatic-like ribonucleases, shows an high selective toxicity to tumor cells1. The native enzyme is an equilibrium mixture of two forms, with (MxM) or without (M=M) swapping of the N-terminal arms2-4. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein5. The cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer1. Indeed this form, despite the breakage of the interchain disulfide bonds, likely to occur in the cytosolic compartment, generates a metastable dimer (NCDBS) with a compact structure that allows the molecule to escape RI6. To generate RNases suitable for antitumor treatment in humans we have dimerized two low immunogenic proteins, bovine pancreatic RNase (RNase A) and its human homologue (HP-RNase). To achieve this, two different strategies have been used. First, we have introduced few selected structural features of the seminal enzyme in these RNases. Differently from NCD-BS, the crystal structure analysis of the NCD form of the RNase A mutants has revealed that they adopt an opened quaternary structure, which can be easily sequestered by RI7. All the dimeric variants, except one, do exhibit a cytotoxic activity at a much lower level than BS-RNase. On the contrary, the dimeric HP-RNase variant (HHP2-RNase), in which the putative swapping determinants of the seminal enzyme have been introduced, was found to be extremely cytotoxic8. Surprisingly, this activity is associated also with the unswapped form of the protein. The crystal structure of this mutant has provided a reasonable explanation for this result9. Secondly, in order to force the swapping in HP-RNase, we have deleted five residues in the loop connecting the N-terminal helix to the core of the protein (desHP-RNase). This last variant is enzymatically active10 and has a very low cytotoxic action. The crystal structure of desHP-RNase reveals a tetrameric association of two swapped dimers with a quaternary assembly compatible with RI binding. Interestingly, the analysis of packing interactions suggests a pathway of large-scale oligomerization. On this basis, fibril formation was predicted and observed.