Interactions of GroEL/GroES with a Heterodimeric Intermediate during α2β2 Assembly of Mitochondrial Branched-chain α-Ketoacid Dehydrogenase: cis CAPPING OF THE NATIVE-LIKE 86-kDa INTERMEDIATE BY GroES

Jiu-Li Song,R.Max Wynn,David T Chuang

doi:10.1074/jbc.m002038200

Jiu-Li Song, R.Max Wynn + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m002038200

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Abstract

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.

Highlights

It has been established that a significant number of proteins depend on the function of pre-existing protein machinery, i.e. molecular chaperones to promote proper folding in an energydependent manner [1,2,3]
The folding of proteins in the GroEL cavity is driven by Mg-ATP hydrolysis in the equatorial domain of the cis ring
To further delineate the nature of this interaction, we determined the amount of GroEL required for the efficient reconstitution of BCKD with the ␣␤ heterodimeric intermediate as a substrate

Summary

Introduction

It has been established that a significant number of proteins depend on the function of pre-existing protein machinery, i.e. molecular chaperones to promote proper folding in an energydependent manner [1,2,3]. Incubation of the GroEL-␣␤ complex with Mg-ATP and GroES resulted in dissociation and discharge of both the ␣ and ␤ subunits from GroEL.

Results

Conclusion