Comparison of the structural and functional properties of RNase A and BS-RNase: a stepwise mutagenesis approach.
The original structure of bovine seminal ribonuclease (BS-RNase), solved in 1993, represents a milestone in the story of protein structure, because it represented the first X-ray structure showing two polypeptide chains entangled through their terminal regions. It is generally assumed that this structural feature is the basis of several special biological activities, including a potent antitumor activity, but this has not been yet definitely proved. To assess this hypothesis, in this article we have analyzed the effects of the N-terminal hinge region and/or of Arg80 on the swapping propensity and cytotoxicity in newly designed proteins, using a covalent dimeric variant of bovine pancreatic ribonuclease (RNase A) as scaffold. All the proteins have a very poor cytotoxic activity, independently on the swapping propensity, that can even reach the same value of native BS-RNase. Overall our data suggest that the swapping represents still an essential requisite for the cytotoxic activity, because it keeps the dimeric structure stable even in the reducing cytosolic environment, but other features are essential to design dimeric antitumor ribonucleases, including a strong positive potential at the N-terminal face and a quaternary structure able to evade the cytosolic ribonuclease inhibitor, with or without the interchain disulfide bridges.
Ercole
Carmine
C
Dipartimento di Chimica, Università degli Studi di Napoli, Via Cintia, Napoli 80126, Italy.
Colamarino
Rosa Angela
RA
Pizzo
Elio
E
Fogolari
Federico
F
Spadaccini
Roberta
R
Picone
Delia
D
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; C013241:ribonuclease dimer; D004722:Endoribonucleases; D012260:Ribonucleases; C032523:ribonuclease SPL; D012259:Ribonuclease, Pancreatic