Domain Structure and Denaturation of a Dimeric Mip-like Peptidyl-Prolyl cis–trans Isomerase from Escherichia coli

Abstract

FKBP22, a protein expressed by Escherichia coli, possesses PPIase (peptidyl-prolyl cis-trans isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with Legionella Mip (a virulence factor) and its related proteins. To understand the domain structure and the folding-unfolding mechanism of Mip-like proteins, we investigated a recombinant E. coli FKBP22 (His-FKBP22) as a model protein. Limited proteolysis indicated that His-FKBP22 harbors an N-terminal domain (NTD), a C-terminal domain (CTD), and a long flexible region linking the two domains. His-FKBP22, NTD(+) (NTD with the entire flexible region), and CTD(+) (CTD with a truncated flexible region) were unfolded by a two-state mechanism in the presence of urea. Urea induced the swelling of dimeric His-FKBP22 molecules at the pretransition state but dissociated it at the early transition state. In contrast, guanidine hydrochloride (GdnCl)-induced equilibrium unfolding of His-FKBP22 or NTD(+) and CTD(+) seemed to follow three-step and two-step mechanisms, respectively. Interestingly, the intermediate formed during the unfolding of His-FKBP22 with GdnCl was not a molten globule but was thought to be composed of the partially unfolded dimeric as well as various multimeric His-FKBP22 molecules. Dimeric His-FKBP22 did not dissociate gradually with increasing concentrations of GdnCl. Very low GdnCl concentrations also had little effect on the molecular dimensions of His-FKBP22. Unfolding with either denaturant was found to be reversible, as refolding of the unfolded His-FKBP22 completely, or nearly completely, restored the structure and function of the protein. Additionally, denaturation of His-FKBP22 appeared to begin at the CTD(+).

Inhibitor-Induced Conformational Stabilization and Structural Alteration of a Mip-Like Peptidyl Prolyl cis-trans Isomerase and Its C-Terminal Domain

“…The tertiary structure of isolated CTD + appears to be a little different than that of the C-terminal domain in FKBP22 [12]. This domain was also reported to be less stable than both rFKBP22 and NTD + (NTD of E. coli FKBP22 with a long hinge region).…”

“…Rapamycin and FK506 but not the juglone or cyclosporin A inhibited the PPIase (peptidyl-prolyl cis-trans isomerase; EC 5.1.2.8) activity of the Mip proteins and their orthologs [11]. Structural investigations suggested a V-shaped structure for the dimeric Mip-like proteins [12]–[15]. Each dumbbell-shaped monomer is composed of a C-terminal domain (CTD), a hinge region, and an N-terminal domain (NTD).…”

“…This domain was also reported to be less stable than both rFKBP22 and NTD + (NTD of E. coli FKBP22 with a long hinge region). Unlike FKBP22, GdnCl-induced denaturation of CTD + followed a two-state mechanism [12]. Despite the altered structure, CTD + bound rapamycin nearly similarly to that of a recombinant FKBP22 [12].…”

“…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. Remarkably, 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].…”

“…Unlike FKBP22, GdnCl-induced denaturation of CTD + followed a two-state mechanism [12]. Despite the altered structure, CTD + bound rapamycin nearly similarly to that of a recombinant FKBP22 [12]. Proteins, which are orthologous to CTD + , exhibited PPIase activity with the peptide substrates as well [16], [29], [32].…”