Stability of the two wings of the coiled-coil domain of ClpB chaperone is critical for its disaggregation activity.
The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in co-operation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA (ATPase associated with various cellular activities)-1 and AAA-2, and an 85-A (1 A=0.1 nm)-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between leucine and isoleucine residues of two amphipathic alpha-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with alanine. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chaperone activities, were able to exert the threading of soluble denatured proteins through their central pore. The destabilization of wing-1, but not wing-2, resulted in a several-fold stimulation of ATPase activity. These results indicate that stability of both wings of the coiled-coil is critical for full functioning of ClpB, but not for the central-pore threading of substrate proteins, and that wing-1 is involved in the communication between AAA-1 and AAA-2.
Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto 8-9-1, Kobe 658-8501, Japan. ywatanab@center.konan-u.ac.jp
Yo-Hei
YH
Watanabe
Yosuke
Y
Nakazaki
Ryoji
R
Suno
Masasuke
M
Yoshida
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002364:Caseins; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016963:Thermus thermophilus / Q000378:metabolism
D001426:Bacterial Proteins; D002364:Caseins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D021122:Protein Subunits