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 cons...

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Published inBiochemical journal Vol. 421; no. 1; p. 71
Main Authors Watanabe, Yo-Hei, Nakazaki, Yosuke, Suno, Ryoji, Yoshida, Masasuke
Format Journal Article
LanguageEnglish
Published England 01.07.2009
Subjects
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Caseins
Gene Expression Regulation, Bacterial - physiology
Heat-Shock Proteins - genetics
Heat-Shock Proteins - metabolism
Models, Molecular
Molecular Chaperones
Mutation
Protein Conformation
Protein Denaturation
Protein Structure, Tertiary
Protein Subunits
Thermus thermophilus - metabolism
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Summary: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.
ISSN:1470-8728
DOI:10.1042/bj20082238