Mechanism of regulation of Hsp70 chaperones by DnaJ cochaperones
Mechanism of regulation of Hsp70 chaperones by DnaJ cochaperones
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DOI：
10.1073/pnas.96.10.5452
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发表时间：
1999-05-11
影响因子：
11.1
通讯作者：
Bukau, B
中科院分区：
文献类型：
--
作者：
Laufen, T;Mayer, MP;Bukau, B
Hsp70分子伴侣在蛋白质的整个生命周期内协助多种蛋白质折叠过程。这些活动的核心是Hsp70 bg DnaJ辅伴侣的调节。DnaJ刺激Hsp70水解ATP，这是关闭其底物结合腔并因此允许稳定结合底物的关键步骤。我们发现，DnaJ刺激ATP水解大肠杆菌Hsp70，DnaK，非常有效地超过1000倍，但只有当存在于高，相反，DnaK在荧光素酶重折叠中的伴侣活性在低几百倍的DnaJ浓度下最大。然而，DnaJ即使在这种低浓度下也能够最大地刺激DnaK ATP酶，条件是存在蛋白质底物，表明DnaJ和底物的协同作用。肽底物在这种协同作用中效果不佳。DnaJ的行动需要结合蛋白质底物的DnaK的底物结合腔的中央疏水口袋证明了DnaJ的能力降低，刺激ATP水解的DnaK突变体与底物结合的缺陷。在高浓度下，DnaJ本身在被认为是非生理的过程中充当DnaK的底物。突变体分析进一步揭示，DnaJ介导的ATP水解的刺激需要DnaK的ATP酶和底物结合结构域之间的通信。因此，这种机制允许DnaJ将DnaK的ATP水解与底物结合紧密偶联，并避免DnaK分子伴侣被肽堵塞。它可能是保守的热休克蛋白70家族成员，并建议占他们的功能多样性。
Hsp70 chaperones assist a large variety of protein folding processes within the entire lifespan of proteins. Central to these activities is the regulation of Hsp70 bg DnaJ cochaperones. DnaJ stimulates Hsp70 to hydrolyze ATP, a key step that closes its substrate-binding cavity and thus allows stable binding of substrate. We show that DnaJ stimulates ATP hydrolysis by Escherichia coli Hsp70, DnaK, very efficiently to >1000-fold, but only if present at high (micromolar) concentration, In contrast, the chaperone activity of DnaK in luciferase refolding was maximal at several hundredfold lower concentration of DnaJ, However, DnaJ was capable of maximally stimulating the DnaK ATPase even at this low concentration, provided that protein substrate was present, indicating synergistic action of DnaJ and substrate. Peptide substrates were poorly effective in this synergistic action. DnaJ action required binding of protein substrates to the central hydrophobic pocket of the substrate-binding cavity of DnaK as evidenced by the reduced ability of DnaJ to stimulate ATP hydrolysis by a DnaK mutant with defects in substrate binding. At high concentrations, DnaJ itself served as substrate for DnaK in a process considered to be unphysiological, Mutant analysis furthermore revealed that DnaJ-mediated stimulation of ATP hydrolysis requires communication between the ATPase and substrate-binding domains of DnaK. This mechanism thus allows DnaJ to tightly couple ATP hydrolysis by DnaK with substrate binding and to avoid jamming of the DnaK chaperone with peptides. It probably is conserved among Hsp70 family members and is proposed to account for their functional diversity.