Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

Abstract

The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 '-deoxycytidylyl(3 ',5 ')-2 '-deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

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

“…In contrast, RNase A dimers are endowed with a limited cytotoxic activity [30] and they are bound by RI [31]. Furthermore, no RNase A dimeric variant displaying a relative subunit orientation that enables the evasion of RI binding [16] under the reducing cytosol conditions [32,33], has been yet produced.…”

The multiple forms of bovine seminal ribonuclease: Structure and stability of a C‐terminal swapped dimer

“…The comparison of the swapping properties of RNase A and BS-RNase can provide interesting clues to investigate the 3D-DS mechanism and its functional implications. Despite the high similarity of their amino acid sequence (more than 80% identity) the two proteins have a completely different behaviour with respect to the swapping [12,14,17,26,29]. The N-terminal swapping mechanism in both systems has already been studied [12,14,17,26,29] and the residues playing a key role in the process have been identified [17].…”

“…Studies on dimeric variants of RNase A, obtained by introducing in its sequence residues playing a key role in the acquisition of the BS‐RNase 3D‐structure, have confirmed the strict relation between antitumor activity and NCD‐BS quaternary architecture. Indeed, none of these dimeric variants retains in its non‐covalent form a compact quaternary structure able to evade RI [17]. This result well correlates with the much lower cytotoxic activity of these RNase A variants as compared to BS‐RNase [18].…”

A review on protein oligomerization process

“…The native monomeric enzyme only degrades single-stranded (ss) RNA and is not cytotoxic, while its artificial dimers and oligomers, either forming through non-covalent DS or covalent bonds, also become active against double-stranded (ds) RNA. 32,92,105,177 Moreover, they can become selectively cytotoxic towards cancer cells both in vitro and in vivo, [178][179][180][181] although some results indicate that cytotoxicity can be dependent also on the type of cell line studied. 35 This acquired cytotoxic power can be considered benign, given the selectivity towards malignant cells, and can be mainly ascribed to the possibility of oligomeric RNases to evade the ribonuclease inhibitor (RI), which is designed to tightly trap monomeric RNases.…”