Schlee S, Groemping Y, Herde P, Seidel R, Reinstein J
Abteilung physikalische Biochemie, Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, Dortmund, D-44227, Germany.
J Mol Biol. 2001 Mar 2;306(4):889-99. doi: 10.1006/jmbi.2001.4455.
ClpB belongs to the Hsp100 family and assists de-aggregation of protein aggregates by DnaK chaperone systems. It contains two Walker consensus sequences (or P-Loops) that indicate potential nucleotide binding domains (NBD). Both domains appear to be essential for chaperoning function, since mutation of the conserved lysine residue of the GX(4)GKT consensus sequences to glutamine (K204Q and K601Q) abolishes its properties to accelerate renaturation 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 changed properties of co-operativity of ATPase activity connected to oligomerization properties to form productive oligomers. This view is corroborated by data that show that structural stability (as judged by CD spectroscopy) or 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 (as judged by binding studies with fluorescent nucleotide analogues and competitive displacement titrations) do not differ dramatically. However, the general pattern of formation of stable, defined oligomers formed as a function of salt concentration and nucleotides and more importantly, cooperativity of ATPase activity at high ATP concentrations is dramatically changed with the two P-loop mutants described.
ClpB属于Hsp100家族，可协助DnaK伴侣系统使蛋白质聚集体解聚。它包含两个沃克一致序列（或P环），这表明其具有潜在的核苷酸结合结构域（NBD）。这两个结构域对于伴侣功能似乎都是必不可少的，因为将GX(4)GKT一致序列中保守的赖氨酸残基突变为谷氨酰胺（K204Q和K601Q）会使其加速聚集的萤火虫荧光素酶复性的特性丧失。这种功能故障的潜在生化原因似乎不是任何一个P环本身的ATP酶活性显著降低，而是与形成有活性寡聚体的寡聚化特性相关的ATP酶活性协同性质发生了改变。这一观点得到了以下数据的证实：这些突变对结构稳定性（通过圆二色光谱法判断）或单周转条件下（低ATP浓度）的ATP酶活性没有显著影响。此外，野生型蛋白和突变体的核苷酸结合特性（通过与荧光核苷酸类似物的结合研究和竞争性置换滴定法判断）没有显著差异。然而，随着盐浓度和核苷酸的变化形成稳定、明确的寡聚体的一般模式，更重要的是，上述两个P环突变体在高ATP浓度下ATP酶活性的协同性发生了显著变化。
