ATP Binding to Nucleotide Binding Domain (NBD)1 of the ClpB Chaperone Induces Motion of the Long Coiled-coil, Stabilizes the Hexamer, and Activates NBD2

Yo-Hei Watanabe,Misa Takano,Masasuke Yoshida

doi:10.1074/jbc.m414623200

Yo-Hei Watanabe, Misa Takano + Show 1 more

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https://doi.org/10.1074/jbc.m414623200

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The molecular chaperone ClpB can rescue the heat-damaged proteins from an aggregated state in cooperation with other chaperones. It has two nucleotide binding domains (NBD1 and NBD2) and forms a hexamer ring in a manner dependent on ATP binding to NBD1. In the crystal structure of ClpB with both NBDs filled by nucleotides, the linker between two NBDs forms an 85-A-long coiled-coil that extends on the outside of the hexamer and leans to NBD1. To probe the possible motion of the coiled-coil, we tested the accessibility of a labeling reagent, fluorescence change of a labeled dye, and cross-linking between the coiled-coil and NBD1 by using the mutants with defective NBD1 or NBD2. The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure. This motion results in stabilization of the hexamer form of ClpB and promotion of ATP hydrolysis at NBD2.

Highlights

A molecular chaperone ClpB is unique in its activity to rescue heat-damaged proteins from an aggregated state in cooperation with a trio DnaK chaperone set, DnaK/DnaJ/GrpE
The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure
Because the 2KT/AA mutant retains nucleotide binding ability to NBD1 but not to NBD2, these results indicated that the shift of the wing-2 to a relatively more hydrophilic environment is induced by nucleotide binding mainly to NBD1

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A molecular chaperone ClpB is unique in its activity to rescue heat-damaged proteins from an aggregated state in cooperation with a trio DnaK chaperone set, DnaK/DnaJ/GrpE. The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure. ABD-labeled ClpB mutants (0.1 mg/ml) in 50 mM MOPS-NaOH (pH 7.5), 150 mM KCl, 5 mM MgCl2 in the presence or the absence of nucleotide were preincubated for 3 min in a sealed cuvette at 55 °C, and fluorescence was measured with a Hitachi F4500 fluorometer (excitation 390 nm, emission spectra 400 – 650 nm).

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