Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.
Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.
Ercole
Carmine
C
Dipartimento di Chimica, Università di Napoli Federico II, Italy.
Avitabile
Francesca
F
Del Vecchio
Pompea
P
Crescenzi
Orlando
O
Tancredi
Teodorico
T
Picone
Delia
D
D000495:Allosteric Site; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D019281:Dimerization; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012661:Semen / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D021122:Protein Subunits; D012259:Ribonuclease, Pancreatic