Identification of the region responsible for fibril formation in the CAD domain of caspase-activated DNase.
Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one alpha-helix and five beta-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing alpha-helix at low pH in the presence of salt. To obtain insights into the mechanism of amyloid fibril formation, we identified the peptide region essential for fibril formation of CAD-CD and the region responsible for the salt requirement. We searched for these regions by constructing a series of deletion and point mutants of CAD-CD. Fibril formation by these CAD-CD mutants was examined by fluorescence analysis of thioflavin T and transmission electron microscopy. C-Terminal deletion and point mutation studies revealed that an aromatic residue near the C-terminus (Trp81) is critical for fibril formation. In addition, the main chain conformation of the beta5 strand, which forms a hydrophobic core with Trp81, was found to be important for the fibril formation by CAD-CD. The N-terminal 30 amino acid region containing two beta-strands was not essential for fibril formation. Rather, the N-terminal region was found to be responsible for the requirement of salt for fibril formation.
Nakamura
Tsutomu
T
National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577.
Kobayashi
Atsuko
A
Hagihara
Yoshihisa
Y
Takai
Tomoyo
T
Mine
Shouhei
S
Yamazaki
Toshio
T
Yumoto
Noboru
N
Uegaki
Koichi
K
D000682:Amyloid / Q000096:biosynthesis*; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012492:Salts; D012965:Sodium Chloride
D000682:Amyloid; D012492:Salts; D012965:Sodium Chloride; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease