Abstract
E. coli ClpB is a heat shock protein that belongs to the AAA+ protein family. Studies have shown that ClpB and its eukaryotic homologue, Hsp104, can disaggregate denatured proteins by themselves or cooperate with the DnaK chaperone system in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity and ClpB majorly exist as hexamer in the presence of nucleoside triphosphate. In contrast to this conclusion, our sedimentation velocity data show that ClpB resides in a monomer-dimer-tetramer-hexamer equilibrium in the presence of ATPϒS (a slowly hydrolysable ATP analog). ClpB hexamers exhibit fast subunit exchange in the absence of nucleoside triphosphate, while the exchange rates decrease when the binding of nucleotide approaching to saturation. For the first time, we determined the binding constants and stoichiometries for ATPϒS to each ClpB oligomer. The monomer is only able to bind one nucleotide whereas all twelve sites in the hexameric ring are bound by nucleotide. Interestingly, dimers and tetramers exhibit stoichiometries of ∼3 and 7, respectively, which is one fewer than the maximum number of binding sites, which suggest an open conformation for the intermediates. We also determined the assembly constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to co-chaperones and polypeptide substrates. We anticipate our studies on ClpB assembly to be a starting point for understanding how ClpB hexamers disaggregate protein aggregates.
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