Determination of Regions in the Dihydrofolate Reductase Structure That Interact with the Molecular Chaperonin GroEL

Abstract

Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state. In contrast, murine DHFR shows a strong interaction with GroEL. Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E. coli protein. Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL. On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E. coli DHFR. In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E. coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q. In the second mutant (EcDHFR-i(7)136), residues 136-139 (V-F-S-E) of E. coli DHFR were replaced with the seven amino acid sequence L-P-E-Y-P-G-V. Both E. coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR. The binding was specific since the presence of MgATP caused the release of the proteins from GroEL. As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL. Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins. A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples. The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer. Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM.

Folding intermediates of β‐lactamase recognized by GroEL

“…Non-native forms of pre-ß-lase [10], a carbamidomethylated variant of RNase Tl [19], DHFR [20], rhodanese [21] and a-lactalbumin [22] among others, have been shown to interact and bind tightly to GroEL, although for DHFR these results have not been confirmed by more recent works [23,24]. For the latter three proteins it has been proposed that they are bound in a molten globule state [20,25], while for an antibody Fab fragment a very late folding intermediate was suggested to bind to GroEL [26].…”

GroES in the asymmetric GroEL 14 –GroES 7 complex exchanges via an associative mechanism

“…GroES 7 and GroEL 14 were purified as described previously (19,20). Protein concentrations were determined by the method of Bradford (21).…”

GroEL‐Mediated protein folding

“…An analogous calculation has been made for the complex of E. coli DHFR with GroEL, with an estimate of <85 nM (Clark et al, 1996). The early unfolded intermediates of LDH formed complexes with GroEL with apparent dissociation constants of -7 nM; the last, molten globule-like, folding intermediate of LDH bound less well by a factor of 5-6 (Badcoe et al, 1991;Staniforth et al, 1994a).…”

“…For example, whereas human DHFR is quantitatively recognized and bound by GroEL during its refolding in vitro, the E. coli protein, which has a virtually identical fold but only -30% identity of amino acid sequence, is not recognized at all (Clark et al, 1996). The refolding human protein is highly aggregation-prone in the absence of GroEL, whereas the refolding E. coli protein is remarkably soluble, even at high concentrations (MS. Goldberg & A.L.…”