Song J L, Wynn R M, Chuang D T
Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
J Biol Chem. 2000 Jul 21;275(29):22305-12. doi: 10.1074/jbc.M002038200.
We showed previously that the interaction of an alphabeta heterodimeric intermediate with GroEL/GroES is essential for efficient alpha(2)beta(2) assembly of human mitochondrial branched-chain alpha-ketoacid dehydrogenase. In the present study, we further characterized the mode of interaction between the chaperonins and the native-like alphabeta heterodimer. The alphabeta heterodimer, as an intact entity, was found to bind to GroEL at a 1:1 stoichiometry with a K(D) of 1.1 x 10(-)(7) m. The 1:1 molar ratio of the GroEL-alphabeta complex was confirmed by the ability of the complex to bind a stoichiometric amount of denatured lysozyme in the trans cavity. Surprisingly, in the presence of Mg-ADP, GroES was able to cap the GroEL-alphabeta complex in cis, despite the size of 86 kDa of the heterodimer (with a His(6) tag and a linker). Incubation of the GroEL-alphabeta complex with Mg-ATP, but not AMP-PNP, resulted in the release of alpha monomers. In the presence of Mg-ATP, the beta subunit was also released but was unable to assemble with the alpha subunit, and rebound to GroEL. The apparent differential subunit release from GroEL is explained, in part, by the significantly higher binding affinity of the beta subunit (K(D) < 4.15 x 10(-9)m) than the alpha (K(D) = 1.6 x 10(-8)m) for GroEL. Incubation of the GroEL-alphabeta complex with Mg-ATP and GroES resulted in dissociation and discharge of both the alpha and beta subunits from GroEL. The beta subunit upon binding to GroEL underwent further folding in the cis cavity sequestered by GroES. This step rendered the beta subunit competent for reassociation with the soluble alpha subunit to produce a new heterodimer. We propose that this mechanism is responsible for the iterative annealing of the kinetically trapped heterodimeric intermediate, leading to an efficient alpha(2)beta(2) assembly of human branched-chain alpha-ketoacid dehydrogenase.
我们之前表明，αβ异二聚体中间体与GroEL/GroES的相互作用对于人线粒体支链α-酮酸脱氢酶高效组装成α(2)β(2)至关重要。在本研究中，我们进一步表征了伴侣蛋白与天然样αβ异二聚体之间的相互作用模式。发现αβ异二聚体作为一个完整实体，以1:1的化学计量比与GroEL结合，解离常数（K(D)）为1.1×10⁻⁷ m。GroEL-αβ复合物的1:1摩尔比通过该复合物在反式腔中结合化学计量的变性溶菌酶的能力得到证实。令人惊讶的是，在Mg-ADP存在下，尽管异二聚体大小为86 kDa（带有His(6)标签和接头），GroES仍能够在顺式方向封闭GroEL-αβ复合物。将GroEL-αβ复合物与Mg-ATP而非AMP-PNP一起温育，导致α单体释放。在Mg-ATP存在下，β亚基也被释放，但无法与α亚基组装，并重新结合到GroEL上。从GroEL释放亚基的明显差异，部分原因是β亚基（K(D) < 4.15×10⁻⁹ m）对GroEL的结合亲和力显著高于α亚基（K(D) = 1.6×10⁻⁸ m）。将GroEL-αβ复合物与Mg-ATP和GroES一起温育，导致α和β亚基从GroEL上解离和释放。β亚基与GroEL结合后，在由GroES隔离的顺式腔中进一步折叠。这一步使β亚基能够与可溶性α亚基重新结合，形成新的异二聚体。我们提出，这种机制负责动力学捕获的异二聚体中间体的迭代退火，从而导致人支链α-酮酸脱氢酶高效组装成α(2)β(2)。
