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

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Abstract

We showed previously that the interaction of an αβ heterodimeric intermediate with GroEL/GroES is essential for efficient α2β2 assembly of human mitochondrial branched-chain α-ketoacid dehydrogenase. In the present study, we further characterized the mode of interaction between the chaperonins and the native-like αβ heterodimer. The αβ 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-αβ 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 MgADP, GroES was able to cap the GroEL-αβ complex in cis, despite the size of 86 kDa of the heterodimer (with a His6 tag and a linker). Incubation of the GroEL-αβ complex with Mg-ATP, but not AMP-PNP, resulted in the release of α monomers. In the presence of Mg-ATP, the β subunit was also released but was unable to assemble with the α subunit, and rebound to GroEL. The apparent differential subunit release from GroEL is explained, in part, by the significantly higher binding affinity of the β subunit (K(D) < 4.15 x 10-9 M) than the α (K(D) = 1.6 x 10-8 M) for GroEL. Incubation of the GroEL-αβ complex with Mg-ATP and GroES resulted in dissociation and discharge of both the α and β subunits from GroEL. The β subunit upon binding to GroEL underwent further folding in the cis cavity sequestered by GroES. This step rendered the β subunit competent for reassociation with the soluble α 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 α2β2 assembly of human branched-chain α-ketoacid dehydrogenase.

Original languageEnglish (US)
Pages (from-to)22305-22312
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number29
DOIs
StatePublished - Jul 21 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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