Abstract
ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.
Highlights
ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors
As a member of an expanded superfamily of ATPases associated with diverse cellular activities (AAAϩ), ClpB/Hsp104 contains two AAAϩ modules, AAA1 and AAA2, in a polypeptide and forms a hexameric ring-like structure, in which each AAAϩ module constitutes each layer of a two layered ring [8]
In the recent cryo-electron microscopy (cryo-EM) structure of Hsp104 from Saccharomyces cerevisiae (ScHsp104), Glu-401 (Glu-412 of ScHsp104) was found to interact with Arg-180 (Arg-194 of ScHsp104), and the substitution of this residue to Lys caused a loss of chaperone activity [15]
Summary
ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp, and its co-factors. In the high-affinity conformation, the NBD can bind to the edge of MD motif-2 of ClpB/Hsp104 [21, 26] The role of MD motif-2 in binding DnaK NBD is clear, the roles of motif-1 should be investigated to understand the mechanisms involved in controlling MD conformation, and in the regulation of ClpB activity by the MD. To clarify these issues, here we identified functionally important residues in motif-1 and their targets for interaction.
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