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

FKBP22, a protein expressed by <i>Escherichia coli</i>, possesses PPIase (peptidyl-prolyl <i>cis</i>-<i>trans</i> isomerase) activity, binds FK506 (an immunosuppressive drug), and shares homology with <i>Legionella</i> 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 <i>E. coli</i> 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⁺