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

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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.