High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation.
We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.
Zerovnik
Eva
E
Department of Biochemistry and Molecular Biology, JoZef Stefan Institute, Ljubljana, Slovenia.
Skerget
Katja
K
Tusek-Znidaric
Magda
M
Loeschner
Corina
C
Brazier
Marcus W
MW
Brown
David R
DR
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D052160:Benzothiazoles; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000648:ultrastructure; D009154:Mutation; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013844:Thiazoles
D000682:Amyloid; D052160:Benzothiazoles; C524994:CSTB protein, human; D015891:Cystatins; D046912:Multiprotein Complexes; D020033:Protein Isoforms; D013844:Thiazoles; C009462:thioflavin T; D003300:Copper; D055313:Cystatin B