Peptidyl−Prolyl Cis−Trans Isomerase of Bacillus subtilis:  Identification of Residues Involved in Cyclosporin A Affinity and Catalytic Efficiency

Abstract

The 17-kDa peptidyl-prolyl cis-trans-isomerase from Bacillus subtilis (PPiB) is a member of the cyclophilin family and shows strong homology to PPIases of eukaryotic origin (40%) and less identify to PPIase sequences of Gram-negative bacteria (27-32%). Although the majority of residues that form the PPIase active site are highly conserved, three residues, V52, H90, and H109 in the sequence of the B.subtilis PPIase, were found to differ from the sequences found in human (hCyP) and Escherichia coli (eCyP). Also, the binding affinity of cyclosporin A (CsA) to the different PPIases varies in IC(50) values from 6 nM for human PPIase hCyPA and 84 nM for the human hCyPB to over 120 nM for B. subtilis and 3000 nM for E. coli. In addition, a variety of k(cat)/K(m) values, ranging from 1.1 mM(-1) s(-1) for the B. subtilis PPIase to over 10 mM(-1) s(-1) for human and 13 mM(-1) s(-1) for E. coli, were detected using the common substrate suc-Ala-Ala-Pro-Phe-pNA. Through site-specific mutagenesis we demonstrate that the differences in the three mentioned residues are mainly responsible for the variations in IC(50) and k(cat)/K(m) values. Replacement of H90 to N90, or H109 to W109, resembling the amino acid sequence of human hCyPA, resulted in more efficient CsA binding (IC(50) value for H90N, 60 nM, and for H109W, 95 nm), whereas replacement of H90 to R90, or H109 to F109, resembling the amino acid sequence of E. coli eCyP, resulted in less efficient CsA binding (IC(50) value for H90R, 2000nM, and for H109F, 5000 nM). In addition to lower CsA affinity, mutant protein H109F shows a k(cat)/K(m) value of 10.5 mM(-1) s(-1), comparably high to that of the wild-type E. coli protein. In contrast, other mutants like C57F, H90N, H90R, and H109W do not differ significantly in k(cat)/K(m) values from wild-type PPiB. Replacement of V52 to M52, which is conserved in E. coli and all known eukaryotic PPIases, does not show any effect in CsA binding affinity (IC(50) value for V52M, 120 nM), but it raises the catalytic efficiency by 12-fold to k(cat)/K(m) of 14 mM(-1) s(-1). In conclusion, our studies suggest that the unique histidine residues H90 and H109 in B. subtilis PPIase are, at least in part, responsible for its intermediate CsA affinity and that the v52 residue confers the low conversion rate.

Enzyme Dynamics During Catalysis

“…CypA operates in numerous biological processes (16,17). It is the receptor for the immunosuppressive drug cyclosporin A, is essential for HIV infectivity, and accelerates protein folding in vitro by catalyzing the rate-limiting cis/trans isomerization of prolyl peptide bonds (18,19). However, its function in vivo and its molecular mechanism are still in dispute.…”

Cyclophilins: Proteins in search of function

“…There is good evidence now that during slow isomerisation steps in protein folding, cyclophilins stabilize the cis - trans transition state and accelerate isomerisation (Fig. 1), a process that is considered important not only in protein folding but also during the assembly of multidomain proteins 38 . Protein folding requires the assistance of both foldases and molecular chaperones and, in some cases cyclophilins serve in both capacities in the maintenance or assembly of supermolecular complexes.…”

Archaeal peptidyl prolyl cis-trans isomerases PPIases

“…Most of the small CyPs in Eukarya have high binding affinity to CsA; i.e., IC 50 of human CyP18 against CsA is approximately 6 nM (11). Bacterial CyPs have lower affinities for CsA; 2 CyPs from E. coli are insensitive to CsA (12) but Bacillus subtilis BsCyP17 is moderately sensitive (IC 50 =120 nM) (13). Crystallographic analysis has revealed that human HsCyP18 has 8 antiparalell alphasheets with 2 short alpha-helices forming a beta-barrel structure (14).…”