Abstract
Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an MBP-alpha. GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES.
Highlights
Molecular chaperones are a class of proteins which assist folding of other proteins in the cell by preventing or reversing aggregation caused by off-pathway folding reactions [1,2,3]
Unfolded subunit polypeptides are imported into mitochondria as aided by Hsp70 family chaperones, and the final stage of folding and assembly of mitochondrial oligomeric proteins is promoted by chaperonins Hsp60/Hsp10 [11,12,13]
Reconstitution of maltose-binding protein (MBP)-E1 with GroEL/GroES/Mg2ϩ-ATP in vitro provides a novel system to study the question of differential domain folding in the presence of chaperonins
Summary
Heat shock proteins; CDTA, trans1,2-diaminocyclohexane-N,N,NЈ,NЈ-tetraacetic acid; E1, branchedchain ␣-ketoacid decarboxylase; E2, dihydrolipoyl transacylase; E3, dihydrolipoyl dehydrogenase; MBP, maltose-binding protein; MBP-␣, the fusion polypeptide between MBP and the ␣ subunit of E1; Ni-NTA, Ni-nitrilotriacetic acid; PK, proteinase K; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis. Binding of GroES, induces a large conformational change in GroEL, leading to an approximate doubling of the volume in the central cavity of that ring This allows the accommodation of polypeptides of ϳ70 kDa in size [30]. This is in contrast to that observed in the ␣1⁄7GroEL complex where GroES binds to GroEL in both cis and trans configurations These findings provide a paradigm for independent domain folding during chaperonin-assisted folding of large multidomain polypeptides
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