Akoev Vladimir, Gogol Edward P, Barnett Micheal E, Zolkiewski Michal
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Protein Sci. 2004 Mar;13(3):567-74. doi: 10.1110/ps.03422604.
ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA(+) superfamily of ATPases associated with various cellular activities. The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, heptamers, and intermediate-size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATP gamma S, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring-type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATP gamma S. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self-association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA(+) proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA(+) ATPases detected both six- and sevenfold particle symmetry.
ClpB是细菌蛋白质解聚伴侣机制的成员，属于与各种细胞活动相关的AAA(+)ATP酶超家族。ClpB协助强烈聚集的蛋白质重新激活的机制尚不清楚，其寡聚状态也一直存在争议。沉降平衡和沉降速度表明，在生理离子强度且无核苷酸的情况下，来自大肠杆菌的ClpB会发生可逆的自我缔合，涉及单体、七聚体和中等大小寡聚体的蛋白质浓度依赖性群体。在低离子强度条件下，七聚体成为ClpB的主要形式。相反，不可水解的ATP类似物ATPγS以及ADP会稳定六聚体ClpB。一致地，电子显微镜显示，无核苷酸时ClpB的环状寡聚体比存在ATPγS时的更大。因此，不水解ATP的核苷酸结合会使ClpB的自我缔合平衡发生显著变化：从支持七聚体形成的反应转变为支持六聚体形成的反应。我们的结果表明ClpB以及可能其他相关的AAA(+)蛋白如何将核苷酸结合转化为主要的结构转变，并有助于解释为什么之前发表的一些AAA(+)ATP酶的电子显微照片检测到了六倍和七倍的粒子对称性。
