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.
"Conserved tyrosine residues in axial channel loops are strongly influenced by nucleotide binding to NBD2 and are crucial for disaggregation in E. coli and S. cerevisiae (Lum et al., 2004; Schlieker et al., 2004; Weibezahn et al., 2004). ClpB function also depends on the mobility of the coiled coil suggesting the coiled coil either adds a mechanical feature to the translocation process or supplies an additional activity to the complex (Lee et al., 2003; Watanabe et al., 2005). However, the mechanism of translocation and the role of the coiled coil, proposed to be on the exterior of the complex, are not explained by the current structural model (Lee et al., 2003, 2007). "
[Show abstract][Hide abstract] ABSTRACT: Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.
"Being very sensitive to its microenvironment, the ABD-Cys fluorescence is expected to decrease in intensity in hydrophilic environment (Watanabe et al., 2005). A previously constructed mutant XpsE(Cys – , L39C), in which all endogenous cysteines were replaced and the residue L39 was mutated to cysteine, was shown to be functional in secretion (N.-T. "
[Show abstract][Hide abstract] ABSTRACT: Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant XpsE(R286A) is non-functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co-ordinating the N- and C-domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsL(N). As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system.
"The identification of an AAA-1/M domain helix 3 interaction is distinct to previously reported data on the relative positioning of the M domain. M domain localization was largely based on the identification of contacts between the first AAA domain and the upper end of M domain helix 2 that is located opposite to helix 3 (Figure 1) (Lee et al., 2003; Watanabe et al., 2005). Our data reveal that the mobility of the M domain is much greater than initially envisioned from the crystal structure of T. thermophilus ClpB and that the original hexameric ClpB model requires modification. "
[Show abstract][Hide abstract] ABSTRACT: The AAA(+) chaperone ClpB mediates the reactivation of aggregated proteins in cooperation with the DnaK chaperone system. ClpB consists of two AAA domains that drive the ATP-dependent threading of substrates through a central translocation channel. Its unique middle (M) domain forms a coiled-coil structure that laterally protrudes from the ClpB ring and is essential for aggregate solubilization. Here, we demonstrate that the conserved helix 3 of the M domain is specifically required for the DnaK-dependent shuffling of aggregated proteins, but not of soluble denatured substrates, to the pore entrance of the ClpB translocation channel. Helix 3 exhibits nucleotide-driven conformational changes possibly involving a transition between folded and unfolded states. This molecular switch controls the ClpB ATPase cycle by contacting the first ATPase domain and establishes the M domain as a regulatory device that acts in the disaggregation process by coupling the threading motor of ClpB with the DnaK chaperone activity.
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