The Helix Located between the Two Domains of a Mip-like Peptidyl-Prolyl cis-trans Isomerase Is Crucial for Its Structure, Stability, and Protein Folding Ability

FKBP22, a PPIase (peptidyl-prolyl cis-trans isomerase) produced by Escherichia coli, binds FK506 and rapamycin (both immunosuppressive drugs), shares significant homology with the Mip-like virulence factors, and has been thought to carry a long a-helix (namely alpha 3) between its two domains. To understand whether the length of helix alpha 3 plays any role in the structure, function, and stability of FKBP22-like proteins, we studied a recombinant E. coli FKBP22 (rFKBP22) and its four helix alpha 3 mutant variants by various in vitro probes. Of the helix alpha 3 mutants, two were deletion mutants (rFKBP22D5 and rFKBP22D30), whereas the two others were insertion mutants (rFKBP2213 and rFKBP2216). Our investigations revealed that the molecular dimensions, dimerization efficiencies, secondary structures, tertiary structures, stabilities, and protein folding abilities of all mutant proteins are different from those of rFKBP22. Conversely, the rapamycin binding affinities of the mutant proteins were affected very little. Urea-induced unfolding of each protein followed a two-state mechanism and was reversible in nature. Interestingly, rFKBP22D30 was the least stable, whereas rFKBP22I3 appeared to be the most stable of the five proteins. The data together suggest that length of helix alpha 3 contributes significantly to the preservation of the structure, function, and stability of E. coli FKBP22.