© 2005 The Japanese Biochemical Society.
Regular Paper |
Identification of the Region Responsible for Fibril Formation in the CAD Domain of Caspase-Activated DNase
1 National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577; and 2 RIKEN Genomic Science Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045
To whom correspondence should be addressed. National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577. Phone: +81-72-751-9526, Fax: 81-72-751-9628, E-mail: k-uegaki{at}aist.go.jp
Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one 
-helix and five ß-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing -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 ß5 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 ß-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.
* Present address: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871.