Toward an antitumor form of bovine pancreatic ribonuclease: the crystal structure of three noncovalent dimeric mutants.
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.
Merlino
Antonello
A
Department of Chemistry, University of Naples, Complesso Universitario Monte S. Angelo, Via Cinthia, I-80126 Naples, Italy.
Russo Krauss
Irene
I
Perillo
Margherita
M
Mattia
Carlo Andrea
CA
Ercole
Carmine
C
Picone
Delia
D
Vergara
Alessandro
A
Sica
Filomena
F
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D002417:Cattle; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008297:Male; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship
D000970:Antineoplastic Agents; D007527:Isoenzymes; D011994:Recombinant Proteins; C013241:ribonuclease dimer; D011392:Proline; D004722:Endoribonucleases; D012260:Ribonucleases; C032523:ribonuclease SPL; D012259:Ribonuclease, Pancreatic; D007930:Leucine; D003545:Cysteine; D005998:Glycine