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

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.

Identification and characterization of a cyclosporin binding cyclophillin from Staphylococcus aureus Newman

“…The resulting 593 bp DNA fragment was cloned to plasmid pET28a (Novagen) using a standard method [9,10]. The yielded plasmid that carries no mutation in the cloned DNA insert was designated as p1350.…”

“…The cyclosporin A binding affinity (Kd) of rCyp was determined by a procedure as followed to find out the rapamycin binding affinity of a recombinant FKBP22 [10]. …”

“…The p values, standard deviation, and mean were determined as stated [10]. Two results are significant if the related p value is <0.05.…”

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

“…The PPIase activity of rCyp (120 nM) was evaluated by RNase T1 (ribonuclease T1) refolding assay as reported [29, 42]. Previously, our modeling study indicated that one S. aureus Cyp molecule binds to one molecule of CsA [42].…”

“…The unfolding of rCyp and rCyp-CsA were also monitored by a standard transverse urea gradient gel electrophoresis (TUGE) with minor modifications [29, 51, 61]. Briefly, a gel having a 10 - 7% acrylamide gradient and a 0 - 8 M urea gradient was made using a 10% acrylamide solution and a 7% acrylamide solution containing 8 M urea.…”