Szabo A, Langer T, Schröder H, Flanagan J, Bukau B, Hartl F U
Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10345-9. doi: 10.1073/pnas.91.22.10345.
Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.
Hsp70家族的分子伴侣可结合未折叠的多肽链，被认为参与了许多蛋白质的细胞折叠途径。大肠杆菌的Hsp70蛋白DnaK受伴侣蛋白DnaJ和辅因子GrpE调控。为了从生物学角度深入理解Hsp70的作用机制，我们分析了一个模型反应，其中DnaK、DnaJ和GrpE介导变性的萤火虫荧光素酶的折叠。底物蛋白折叠过程中的结合与释放涉及以下依赖ATP水解的循环：（i）未折叠的荧光素酶最初与DnaJ结合；（ii）与荧光素酶-DnaJ相互作用后，DnaK水解其结合的ATP，形成稳定的荧光素酶-DnaK-DnaJ复合物；（iii）GrpE从DnaK上释放ADP；（iv）ATP与DnaK结合触发底物蛋白释放，从而完成反应循环。单次结合与释放循环仅能使一小部分荧光素酶分子折叠。完全高效折叠需要与DnaK和DnaJ进行多轮依赖ATP的相互作用。
