Doyle Shannon M, Hoskins Joel R, Wickner Sue
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11138-44. doi: 10.1073/pnas.0703980104. Epub 2007 Jun 1.
ClpB and Hsp104, members of the AAA+ superfamily of proteins, protect cells from the devastating effects of protein inactivation and aggregation that arise after extreme heat stress. They exist as a hexameric ring and contain two nucleotide-binding sites per monomer. ClpB and Hsp104 are able to dissolve protein aggregates in conjunction with the DnaK/Hsp70 chaperone system, although the roles of the individual chaperones in disaggregation are not well understood. In the absence of the DnaK/Hsp70 system, ClpB and Hsp104 alone are able to perform protein remodeling when their ATPase activity is asymmetrically slowed either by providing a mixture of ATP and ATP gamma S, a nonphysiological and slowly hydrolyzed ATP analog, or by inactivating one of the two nucleotide-binding domains by mutation. To gain insight into the roles of ClpB and the DnaK system in protein remodeling, we tested whether there was a further stimulation by the DnaK chaperone system under conditions that elicited remodeling activity by ClpB alone. Our results demonstrate that ClpB and the DnaK system act synergistically to remodel proteins and dissolve aggregates. The results further show that ATP is required and that both nucleotide-binding sites of ClpB must be able to hydrolyze ATP to permit functional collaboration between ClpB and the DnaK system.
ClpB和Hsp104是AAA+超家族蛋白的成员，可保护细胞免受极端热应激后蛋白质失活和聚集所带来的毁灭性影响。它们以六聚体环的形式存在，每个单体含有两个核苷酸结合位点。ClpB和Hsp104能够与DnaK/Hsp70伴侣系统协同溶解蛋白质聚集体，尽管各个伴侣在解聚过程中的作用尚未完全明确。在缺乏DnaK/Hsp70系统的情况下，当通过提供ATP和ATPγS（一种非生理性且水解缓慢的ATP类似物）的混合物，或者通过突变使两个核苷酸结合结构域之一失活，从而不对称地减缓其ATP酶活性时，ClpB和Hsp104单独就能进行蛋白质重塑。为了深入了解ClpB和DnaK系统在蛋白质重塑中的作用，我们测试了在仅由ClpB引发重塑活性的条件下，DnaK伴侣系统是否会产生进一步的刺激作用。我们的结果表明，ClpB和DnaK系统协同作用以重塑蛋白质并溶解聚集体。结果还进一步表明，需要ATP，并且ClpB的两个核苷酸结合位点都必须能够水解ATP，才能使ClpB和DnaK系统之间进行功能协作。
