Abstract
Cyclophilins, a class of peptidyl-prolyl cis-trans isomerase (PPIase) enzymes, are inhibited by cyclosporin A (CsA), an immunosuppressive drug. Staphylococcus aureus Newman, a pathogenic bacterium, carries a gene for encoding a putative cyclophilin (SaCyp). SaCyp shows significant homology with other cyclophilins at the sequence level. A three-dimensional model structure of SaCyp harbors a binding site for CsA. To verify whether SaCyp possesses both the PPIase activity and the CsA binding ability, we have purified and investigated a recombinant SaCyp (rCyp) using various in vitro tools. Our RNase T1 refolding assay indicates that rCyp has a substantial extent of PPIase activity. rCyp that exists as a monomer in the aqueous solution is truly a cyclophilin as its catalytic activity specifically shows sensitivity to CsA. rCyp appears to bind CsA with a reasonably high affinity. Additional investigations reveal that binding of CsA to rCyp alters its structure and shape to some extent. Both rCyp and rCyp-CsA are unfolded via the formation of at least one intermediate in the presence of guanidine hydrochloride. Unfolding study also indicates that there is substantial extent of thermodynamic stabilization of rCyp in the presence of CsA as well. The data suggest that rCyp may be exploited to screen the new antimicrobial agents in the future.
Highlights
A newly synthesized polypeptide becomes functional only when it is folded correctly
Identification of a putative cyclophilin from S. aureus: To identify whether a cyclophilin-like peptidyl-prolyl cis-trans isomerase (PPIase) is encoded by S. aureus, we have carried out a blastP analysis using the sequence of a B. subtilis PpiB, one of the first discovered cyclophilins from a Gram-positive bacterium [1]
To understand whether the unfolding of SaCyp in the presence and absence of cognate drug occurs by a similar mechanism, we have separately investigated the GdnCl-induced unfolding of recombinant SaCyp (rCyp) and rCyp-cyclosporin A (CsA) by the far-UV circular dichroism (CD) and intrinsic Trp fluorescence spectroscopy
Summary
A newly synthesized polypeptide becomes functional only when it is folded correctly. Many chaperones and isomerases are usually involved in the folding of nascent polypeptides [1]. Enzymes like peptidyl-prolyl cis-trans isomerases (PPIases) stimulate the folding of polypeptides primarily by catalyzing the cis-trans isomerization of peptide bonds preceding the proline residues. Despite the well-defined catalytic activity, currently little is known about the exact cellular substrates of these enzymes. Several reports have demonstrated that PPIases are involved in the variety of cellular functions such as signal transduction, transcriptional regulation, cell differentiation, protein secretion, and apoptosis [1]. The links of PPIases in many diseases have been clearly established
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