Rye H S, Burston S G, Fenton W A, Beechem J M, Xu Z, Sigler P B, Horwich A L
Howard Hughes Medical Institute, Department of Genetics, School of Medicine, Yale University, New Haven, Connecticut 06510, USA.
Nature. 1997 Aug 21;388(6644):792-8. doi: 10.1038/42047.
The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.
伴侣蛋白GroEL是一种双环结构，每个环都有一个中央腔，当在腺嘌呤核苷酸存在的情况下被共伴侣蛋白GroES封闭时，该中央腔为蛋白质的高效折叠提供了一个环境。在顺式三元复合物中观察到了底物硫氰酸酶的有效折叠，其中GroES和多肽与同一环结合，该复合物由ATP、ADP或不可水解的ATP类似物形成，这表明对ATP的特定需求仅限于反式环中的一种作用，即从顺式侧排出GroES和多肽。然而，我们在此表明，对于苹果酸脱氢酶和核酮糖-1,5-二磷酸羧化酶的折叠，顺式环中对ATP也有绝对需求，因为ADP和AMP-PNP无法促进折叠。我们使用结合ATP但水解有缺陷的GroEL突变体形式研究了顺式环和反式环中ATP结合和水解的具体作用。顺式环中ATP和GroES的结合在高度稳定的GroEL-ATP-GroES复合物内部启动了有效折叠。为了排出GroES和多肽，顺式环中的ATP水解是必需的，以形成稳定性降低的GroEL-ADP-GroES复合物，使顺式复合物通过反式环中ATP的结合（不水解）而释放。这些观察结果解释了为什么GroEL作为双环复合物发挥作用。
