Jiu Li Song

GroEL/GroES-dependent Reconstitution of α2β2 Tetramers of Human Mitochondrial Branched Chain α-Ketoacid Decarboxylase OBLIGATORY INTERACTION OF CHAPERONINS WITH AN αβ DIMERIC INTERMEDIATE

The decarboxylase component (E1) of the human mitochondrial branched chain α-ketoacid dehydrogenase multienzyme complex (∼4–5 × 103 kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa α subunits and two 37.8-kDa β subunits. In the present study, His6-tagged E1 α2β2 tetramers (171 kDa) denatured in 8 m urea were competently reconstituted in vitro at 23 °C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 m −1 s−1. Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-α and GroEL-β complexes as combined substrates. However, the β subunit was markedly more prone to misfolding than the α in the absence of GroEL. The α subunit was released as soluble monomers from the GroEL-α complex alone in the presence of GroES and Mg-ATP. In contrast, the β subunit discharged from the GroEL-β complex readily rebound to GroEL when the α subunit was absent. Analysis of the assembly state showed that the His6-α and β subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa αβ dimeric intermediate, which subsequently dimerized to produce the active α2β2tetrameter. The purified αβ dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-αβ ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138m −1 s−1. Dimers were regenerated from the GroEL-αβ complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 α2β2 tetramers.

GroEL/GroES Promote Dissociation/Reassociation Cycles of a Heterodimeric Intermediate during α2β2Protein Assembly ITERATIVE ANNEALING AT THE QUATERNARY STRUCTURE LEVEL

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 min 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 = 480m −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.

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

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 × 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 thetrans cavity. Surprisingly, in the presence of Mg-ADP, GroES was able to cap the GroEL-αβ complex in cis, despite the size of 86 kDa of the heterodimer (with a His6tag 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 × 10− 9 m) than the α (K D = 1.6 × 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 thecis 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.

GroEL Structure at 6 Å Resolution Using Electron Cryomicroscopy and EMAN

We present a reconstruction of native apo-GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at ~6 A resolution.This continues the long history of cryo-EM contributions to GroEL research, for example [1,2,3]. As this was largely a test of the reconstruction methodology, and we wished to avoid any possibility of biasing the results, absolutely no reference was made to the x-ray crystal structure [4] at any point during the actual reconstruction process. To perform this reconstruction, 39,085 particles were selected from 42 micrographs collected on a JEOL 2010F electron cryomicroscope. The resolution achieved in this reconstruction was largely due to new features added to the EMAN [5] software suite. While the overall reconstruction methodology remains unchanged, substantial improvements were made to several specific algorithms. Through improvements to the 2-D particle alignment routines, use of improved similarity criteria in particle classification, and reductions in iterative particle class-alignment, we were able to achieve the presented structure at ~6 A resolution as measured by Fourier Shell Correlation using the 0.5 criterion.