Barnett Micheal E, Zolkiewski Michal
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Biochemistry. 2002 Sep 17;41(37):11277-83. doi: 10.1021/bi026161s.
ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.
ClpB是大肠杆菌中多分子伴侣系统（与DnaK、DnaJ和GrpE一起）的成员，该系统可使高度聚集的蛋白质重新激活。ClpB的序列包含两个ATP结合结构域，每个结构域都含有沃克保守基序。ClpB的N端和C端序列区域不包含已知的功能基序。在本研究中，我们对沃克A基序（Lys212和Lys611）以及ClpB的C端区域（Asp797、Arg815、Arg819和Glu826）内选定的带电荷残基进行了定点诱变。我们发现突变K212T、K611T、D797A、R815A、R819A和E826A对ClpB的二级结构没有显著影响。N端ATP结合位点（K212T）的突变，而非C端ATP结合位点（K611T）的突变，以及C端结构域内的两个突变（R815A和R819A）在无核苷酸的情况下抑制了ClpB的自缔合。在ATP和ADP存在的情况下，也观察到了这些突变体自缔合的缺陷。四个突变体K212T、K611T、R815A和R819A表现出伴侣活性受到抑制，这与其在酪蛋白存在下的低ATP酶活性相关。我们的结果表明，位于亚基间界面的带正电荷氨基酸（这包括N端ATP结合结构域的沃克A基序中的Lys212以及C端结构域中的Arg815和Arg819）参与亚基间盐桥并稳定ClpB寡聚体。有趣的是，我们在C端结构域中鉴定出一个保守残基（Arg819），它不直接参与核苷酸结合，但对ClpB的伴侣活性至关重要。
