Date Published: March 29, 2019
Publisher: Public Library of Science
Author(s): Soham Seal, Soumitra Polley, Subrata Sau, Md. Imtaiyaz Hassan.
Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future.
The cyclophilins (EC: 126.96.36.199) represent a family of highly conserved peptidyl-prolyl cis/trans isomerase (PPIase) enzymes those are expressed by most living organisms, and some giant viruses [1–5]. These proteins control protein folding by catalyzing the trans to cis isomerization of the peptidyl bonds those precede proline residues. These enzymes also influence numerous other cellular processes including protein trafficking, transcription, cell differentiation, apoptosis, protein secretion, T-cell activation, and signal transduction. In addition, these folding catalysts play critical roles in developing cardiovascular diseases, rheumatoid arthritis, viral infections, cancer, diabetes, sepsis, asthma, aging, neurodegenerative diseases, and microbial infections [2, 3, 6–11]. The catalytic activities of the cyclophilins are typically inhibited by cyclosporin A (CsA), a cyclic peptide harboring eleven amino acid residues . A ternary complex, formed by the association of CsA-cyclophilin complex with calcineurin, prevents the dephosphorylation of the transcription factor NF-AT that, in turn, blocks the expression of cytokines from T-lymphocytes [12–14]. The reduction of T-cell activity by CsA has made it extremely useful in clinics, particularly for preventing graft rejection after organ and bone marrow transplantation . However, the severe side effects of CsA have restricted its use  and promoted to develop many CsA analogs with no immunosuppressive activity [2, 10, 11, 16–18]. Some of these CsA analogs though yielded promising results have not been approved yet.
The present study has provided some seminal clues about the folding-unfolding mechanism and the domain structure of rCyp (Fig 1A), a chimeric SaCyp harboring 220 amino acid residues . Our limited proteolysis (Fig 1) and the subsequent analyses (Table 1) have revealed that two rCyp ends carrying residues 1 to 22 and 218 to 220 are only susceptible to three proteolytic enzymes employed in the study. The rCyp region having residues 23 to 218 carries most of the cleavage sites of these enzymes (Fig 1A). The absence of digestion in the internal rCyp region indicates the formation of a domain by the residues 23 to 218. The residue 23 in rCyp corresponds to the C-terminal end residue of its polyhistidine tag, whereas its residue 218, equivalent to the residue 195 of SaCyp (Fig1A), is the C-terminal end residue of the domain. Thus, the single domain structure of SaCyp proposed before on the basis of computational studies  was confirmed by our proteolysis results. However, such single domain structure is not unprecedented as the cyclophilins those have masses nearly similar to that of SaCyp are also shown to carry single domain capable of binding both the substrate and inhibitor [2, 3, 11, 19].
Our investigations have provided invaluable clues about the basic structure and the folding-unfolding mechanism of SaCyp, an S. aureus-encoded cyclophilin involved in pathogenesis. We noted that rCyp, a recombinant SaCyp, is a single-domain protein with a short tail at its C-terminal end. Additionally, rCyp unfolds via the formation of an intermediate in the presence of urea. The rCyp intermediate has the native-protein like structure and also shows little loss of CsA binding activity. The unfolding of the CsA-bound rCyp also similarly occurred in the presence of urea. The stability data of rCyp seems to be applicable in the discovery of new CsA derivatives in the future.