The chaperone function of ClpB from Thermus thermophilus depends on allosteric interactions of its two ATP-binding sites

ClpB belongs to the Hsp100 family and assists de-aggregation of protein agg regates by DnaK chaperone systems. It contains two Walker consensus sequenc es (or P-Loops) that indicate potential nucleotide binding domains (NBD). B oth domains appear to be essential for chaperoning function, since mutation of the conserved lysine residue of the GX(4)GKT consensus sequences to glu tamine (K204Q and K601Q) abolishes its properties to accelerate renaturatio n of aggregated firefly luciferase. The underlying biochemical reason for this malfunction appears not to be a dramatically reduced ATPase activity of either P-loop per se but rather cha nged properties of co-operativity of ATPase activity connected to oligomeri zation properties to form productive oligomers. This view is corroborated b y data that show that structural stability las judged by CD spectroscopy) o r ATPase activity at single turnover conditions (at low ATP concentrations) are not significantly affected by these mutations. In addition nucleotide binding properties of wild-type protein and mutants las judged by binding s tudies with fluorescent nucleotide analogues and competitive displacement t itrations) do not differ dramatically. However, the general pattern of form ation of stable, defined oligomers formed as a function of salt concentrati on and nucleotides and more importantly, cooperativity of ATPase activity a t high Am concentrations is dramatically changed with the two P-loop mutant s described. (C) 2001 Academic Press.