Abstract
FKBP22, an Escherichia coli-encoded PPIase (peptidyl-prolyl cis-trans isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD+ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD+. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD+ could be exploited to screen the rapamycin-like inhibitors in the future.
Highlights
FKBP22, a protein-folding catalyst expressed by Escherichia coli [1], harbors 206 amino acid residues, forms dimers in solution and shares significant identity with Shewanella sp
To understand whether rapamycin binding causes any structural alteration of the C-terminal domain of E. coli FKBP22, we developed the model structures of both CTD+ and CTD+rapamycin complex using standard procedures (See Materials and methods for details)
There is a considerable reduction of the fluorescence intensities of both rFKBP22 and CTD+ in the presence of two folds molar excess of rapamycin
Summary
FKBP22, a protein-folding catalyst expressed by Escherichia coli [1], harbors 206 amino acid residues, forms dimers in solution and shares significant identity with Shewanella sp. Structural investigations suggested a V-shaped structure for the dimeric Mip-like proteins [12,13,14,15]. Each dumbbell-shaped monomer is composed of a C-terminal domain (CTD), a hinge region, and an N-terminal domain (NTD). While the N-terminal domain is responsible for dimerization of the molecule, the C-terminal domain possesses the substrate and the inhibitor binding sites. The V-shaped structure including the hinge region was reported to be critical for the PPIase activity of Mip-like proteins with a protein substrate [15,16,17]. A recent study suggested that GdnCl- and urea-induced denaturation of a chimeric E. coli FKBP22 follow a three-state and a two-state mechanism, respectively. Intermediates produced during the denaturation of a recombinant FKBP22 with GdnCl were not molten globules but believed to be made of different incompletely denatured multimers of this protein [12]
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