FKBP22, an Escherichia coli-specific peptidyl-prolyl cis-trans isomerase, shows substantial homology with the Mip-like virulence factors. Mip-like proteins are homodimeric and possess a V-shaped conformation. Their N-terminal domains form dimers, whereas their C-terminal domains bind protein/peptide substrates and distinct inhibitors such as rapamycin and FK506. Interestingly, the two domains of the Mip-like proteins are separated by a lengthy, protease-susceptible α-helix. To delineate the structural requirement of this domain-connecting region in Mip-like proteins, we have investigated a recombinant FKBP22 (rFKBP22) and its three point mutants I65P, V72P and A82P using different probes. Each mutant harbors a Pro substitution mutation at a distinct location in the hinge region. We report that the three mutants are not only different from each other but also different from rFKBP22 in structure and activity. Unlike rFKBP22, the three mutants were unfolded by a non-two state mechanism in the presence of urea. In addition, the stabilities of the mutants, particularly I65P and V72P, differed considerably from that of rFKBP22. Conversely, the rapamycin binding affinity of no mutant was different from that of rFKBP22. Of the mutants, I65P showed the highest levels of structural/functional loss and dissociated partly in solution. Our computational study indicated a severe collapse of the V-shape in I65P due to the anomalous movement of its C-terminal domains. The α-helical nature of the domain-connecting region is, therefore, critical for the Mip-like proteins.
Keywords: A82P, a FKBP22/rFKBP22 derivative harboring a Ala to Pro change at position 82 in the helix α3; CTD, C-terminal domain of FKBP22; FKBP22, a PPIase from Escherichia coli; Helix α3; I65P, a FKBP22/rFKBP22 variant carrying a Ile to Pro replacement at position 65 in the helix α3; Mip, macrophage infectivity potentiator; Mutation; NTD, N-terminal domain of FKBP22; PPIase, peptidyl-prolyl cis-trans isomerase; Peptidyl-prolyl cis-trans isomerase; Stability; Structure; TUGE, transverse urea gradient gel electrophoresis; V72P, a FKBP22/rFKBP22 variant with a Val to Pro substitution at position 72 in the helix α3; rFKBP22, a polyhistidine-tagged FKBP22.