Formation, structure, and dissociation of the ribonuclease S three-dimensional domain-swapped dimer.
Post-translational events, such as proteolysis, are believed to play essential roles in amyloid formation in vivo. Ribonuclease A forms oligomers by the three-dimensional domain-swapping mechanism. Here, we demonstrate the ability of ribonuclease S, a proteolytically cleaved form of ribonuclease A, to oligomerize efficiently. This unexpected capacity has been investigated to study the effect of proteolysis on oligomerization and amyloid formation. The yield of the RNase S dimer was found to be significantly higher than that of RNase A dimers, which suggests that proteolysis can activate oligomerization via the three-dimensional domain-swapping mechanism. Characterization by chromatography, enzymatic assays, and NMR spectroscopy indicate that the structure of the RNase S dimer is similar to that of the RNase A C-dimer. The RNase S dimer dissociates much more readily than the RNase A C-dimer does. By measuring the dissociation rate as a function of temperature, the activation enthalpy and entropy for RNase S dimer dissociation were found to resemble those for the release of the small fragment (S-peptide) from monomeric RNase S. Excess S-peptide strongly slows RNase S dimer dissociation. These results strongly suggest that S-peptide release is the rate-limiting step of RNase S dimer dissociation.
López-Alonso
Jorge P
JP
Instituto de Química-Física "Rocasolano" CSIC, Serrano 119, E-28006 Madrid, Spain.
Bruix
Marta
M
Font
Josep
J
Ribó
Marc
M
Vilanova
María
M
Rico
Manuel
M
Gotte
Giovanni
G
Libonati
Massimo
M
González
Carlos
C
Laurents
Douglas V
DV
D000682:Amyloid / Q000096:biosynthesis; D000818:Animals; D002417:Cattle; D019281:Dimerization; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism; D011108:Polymers; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D013696:Temperature
D000682:Amyloid; D010446:Peptide Fragments; D011108:Polymers; C060871:ribonuclease A S-peptide; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S