Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

Abstract

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2-RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix-II (Gln28fiLeu, Arg31fiCys, Arg32fiCys, and Asn34fiLys) and to eliminate a negative charge on the surface (Glu111fiGly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well-defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.Keywords: crystal structure; antitumor agents; ribonuclease; 3D-domain swapping; dimers Abbreviations: BSRNase, bovine seminal ribonuclease; des(1-7)HP-RNase, human pancreatic ribonuclease in which the first seven residues have been deleted; HHP-RNase, covalent dimeric variant of human pancreatic ribonuclease in which four residues at the helix-II region are mutated according to BSRNase sequence (Gln28!Leu, Arg31!Cys, Arg32!Cys, and Asn34!Lys); HHP2-RNase, HHP-RNase with an additional mutation (Glu111!Gly); HP-RI, structure of human pancreatic ribonuclease complexed with ribonuclease inhibitor; HP-RNase, human pancreatic ribonuclease; MxM-BSRNase, dimeric form of BSRNase in which the chains swap their N-terminal tails; M¼M-BSRNase, unswapped dimer of BSRNase; M¼M-HHP2, unswapped dimer of HHP2-RNase; NCD-BSRNase, noncovalent swapped form of BSRNase; ONC, Onconase; PDB, Protein Data Bank; PM7, human pancreatic ribonuclease in which residues 1-21 are replaced by the corresponding residues of BSRNase; PM8, N-terminal swapped dimer of a variant of human pancreatic ribonuclease; RI, ribonuclease inhibitor; RMSD, root-mean-square deviation.Grant sponsor: Ministero dell'Istruzione, dell'Università e della Ricerca; Grant number: FIRB RBNE03B8KK and PRIN2007.

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

“…The other two substitutions present in the hinge peptide of the seminal enzyme, E111→G and Y115→S, confer a high conformational flexibility to this region, facilitating the dissociation of the dimer in solution. All these experimental data confirm the important role that in the 3D‐DS phenomenon is played by the hinge region [4,38,39], the only structural element that in a swapping process undergoes a rather large conformational rearrangement.…”

“…Indeed the rmsd between one of the two subunits and the monomeric enzyme (PDB code: http://1N1X) is ∼0.7 Å (rmsd calculated for main chain atoms of residues 1–110 and 117–124). A phosphate ion is bound to each composite catalytic site stabilizing the dimer by mimicking the enzyme substrate, as typically observed in several other ribonuclease structures [12,21,29,37,38].…”

The four cysteines ring motif in proteins

“…40,41 PDZ domains possess similar sequence and fold. This usually consists of six b-strands (namely A-F) and two a-helices [30][31][32][33] and the following five-residue bhairpin (CDSPV) connecting strands B-C. A structural comparison with other PDZ domains sharing high-sequence identity and very similar 3D structure (i.e. pdbcode 1g9o) shows a significant difference just in this region, where other PDZ domains are glycine rich.…”

“…30 This explanation of the cytotoxic activity of the swapped form of the seminal enzyme is consistent with the results of several studies carried out on ad hoc designed mutants, 24,31 of the seminal enzyme as well as on those of homologous monomeric pancreatic RNases. In particular, both bovine 32 and human 33 pancreatic RNase have been transformed into cytotoxic dimeric species by introducing the 4CR motif and other residues essential for the swapping. 32,33 Altogether, these studies have clearly demonstrated that (i) the swapped dimerization confers special properties and abilities only to the predominant native isoform M3M, 19,24,25 (ii) the 4CR moiety does not allow monomerization upon unswapping and, (iii) the domain swapping does not allow monomerization upon 4CR reduction.…”

“…In particular, both bovine 32 and human 33 pancreatic RNase have been transformed into cytotoxic dimeric species by introducing the 4CR motif and other residues essential for the swapping. 32,33 Altogether, these studies have clearly demonstrated that (i) the swapped dimerization confers special properties and abilities only to the predominant native isoform M3M, 19,24,25 (ii) the 4CR moiety does not allow monomerization upon unswapping and, (iii) the domain swapping does not allow monomerization upon 4CR reduction.…”