Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis.
The cystatins were the first amyloidogenic proteins to be shown to oligomerize through a 3D domain swapping mechanism. Here we show that, under conditions leading to the formation of amyloid deposits, the domain-swapped dimer of chicken cystatin further oligomerizes to a tetramer, prior to fibrillization. The tetramer has a very similar circular dichroism and fluorescence signature to the folded monomer and dimer structures, but exhibits some loss of dispersion in the 1H-NMR spectrum. 8-Anilino-1-naphthalene sulfonate fluorescence enhancement indicates an increase in the degree of disorder. While the dimerization reaction is bimolecular and most likely limited by the availability of a predominantly unfolded form of the monomer, the tetramerization reaction is first-order. The tetramer is formed slowly (t(1/2)=six days at 85 degrees C), dimeric cystatin is the precursor to tetramer formation, and thus the rate is limited by structural rearrangement within the dimer. Some higher-order oligomerization events parallel tetramer formation while others follow from the tetrameric form. Thus, the tetramer is a transient intermediate within the pathway of large-scale oligomerization.
Sanders
Anna
A
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Jeremy Craven
C
C
Higgins
Lee D
LD
Giannini
Silva
S
Conroy
Matthew J
MJ
Hounslow
Andrea M
AM
Waltho
Jonathan P
JP
Staniforth
Rosemary A
RA
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D015891:Cystatins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000682:Amyloid; D015891:Cystatins; D011994:Recombinant Proteins