GroEL/GroES promote dissociation/reassociation cycles of a heterodimeric intermediate during αg2β2 protein assembly: Iterative annealing at the quaternary structure level

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Abstract

Whereas the mechanism of GroEL/GroES-mediated protein folding has been extensively studied, the role of these chaperonins in oligomeric protein assembly remains poorly understood. In the present study, we investigated the interaction of the chaperonins with an αβ heterodimeric intermediate during the α2β2 assembly of human mitochondrial branched-chain α-ketoacid dehydrogenase/decarboxylase (BCKD). Incubation of the recombinant His6- tagged BCKD in 400 mM KSCN for 45 rain at 23 °C caused a complete dissociation of the α2β2 heterotetramers into inactive αβ heterodimers. Dilution of the denaturant resulted in a rapid recovery of BCKD independent of the chaperonins GroEL/GroES. Prolonged incubation of BCKD in 400 mM KSCN resulted in the generation of nonproductive or 'bad' heterodimers, which were unable to undergo spontaneous reactivation but capable of binding to GroEL to form a stable GroEL-αβ complex. Incubation of this complex with GroES and Mg-ATP led to the slow reactivation of BCKD with a second-order rate constant k = 480 M-1 s-1. Mixing experiments with radiolabeled and unlabeled protein substrates provided direct evidence that GroEL/GroES promote dissociation and subunit exchange between bad heterodimers. This was accompanied by the transformation of bad heterodimers to their 'good' or productive counterparts. The good heterodimers were capable of spontaneous dimerization to initially form an inactive heterotetrameric species, followed by conversion to active heterotetramers. However, a large fraction of bad heterodimers were regenerated and rebound to GroEL. The cycle was perpetuated until the reconstitution of active BCKD was complete. Our data support the thesis that chaperonins GroEL/GroES mediate iterative annealing of nonproductive assembly intermediates at the quaternary structure level. This step is essential for an efficient subsequent higher order oligomerization.

Original languageEnglish (US)
Pages (from-to)2786-2794
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number4
DOIs
StatePublished - Jan 28 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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