A new mutant of bovine seminal ribonuclease with a reversed swapping propensity.
Bovine seminal ribonuclease (BS-RNase) is made up of two identical subunits bridged through two disulfide bonds. In solution, it exists as a 2:1 equilibrium mixture between two forms, with (MxM) and without swapping (M=M) of the N-terminal arms. The swapping endows BS-RNase with some special biological functions, including antitumor activity, since MxM retains a dimeric structure even under reducing conditions, thus evading the cytosolic ribonuclease inhibitor. To investigate the structural basis of domain swapping in BS-RNase, we have obtained several mutants by replacing selected residues with the corresponding ones of its monomeric counterpart, bovine pancreatic ribonuclease (RNase A). We have already shown that, in contrast with all other cases of swapped proteins, the swapping propensity of BS-RNase does not depend on the specific sequence of the 16-22 hinge loop, which connects the main body to the dislocating arm. In this paper we report the design, the expression, and the structural characterization of two mutants obtained by replacing Arg80 with Ser either in BS-RNase or in the mutant already containing the 16-22 hinge sequence of RNase A. NMR and circular dichroism data indicate that, in the monomeric form of the latter mutant, Ser80 acts as a switch for the conformation of the hinge region. Accordingly, in the dimeric form of the same mutant the MxM:M=M equilibrium ratio is inverted to 1:2. Overall, these data suggest that the presence of Arg80 triggers the swapping of N-terminal ends and plays a relevant role in the stability of the swapped form of BS-RNase.
Ercole
Carmine
C
Dipartimento di Chimica, Università Federico II di Napoli, Via Cintia, 80126 Napoli, Italy.
Spadaccini
Roberta
R
Alfano
Caterina
C
Tancredi
Teodorico
T
Picone
Delia
D
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D019143:Evolution, Molecular; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics; D013696:Temperature
D004722:Endoribonucleases; C032523:ribonuclease SPL; D012259:Ribonuclease, Pancreatic