The ubiquitous cyclophilins belong to peptidyl-prolyl cis/trans isomerases (PPIases; EC 5.2.1.8). They are able to catalyze the cis/trans isomerization about peptidyl-prolyl amide bonds. The mode of action of human cytosolic cyclophilin (Cyp18cy) has been studied on substrate analog tetrapeptide-4-nitroanilides containing the thioxo peptidyl-prolyl bond. Five peptides of the general structure Ala-Xaa-psi [CS-N]-Pro-Phe-NH-Np (Xaa = Gly, Ala, (S)-2-aminobutyric acid, Phe, and Leu) containing the thioxo peptidyl-prolyl bond were synthesized. The kcat values for the chymotryptic cleavage of 4-nitroanilide bond of the thioxo tetrapeptide-4-nitroanilides ranged from 1.7 to 9.0 s-1 and were sufficiently high to analyze the conformational equilibria by isomer-specific proteolysis. The rate constants of the cis/trans isomerization of the thioxo peptidyl-prolyl bond were found to be 25-100-fold lower due to the O/S substitution. Cyp18cy binds both thioxo peptides and oxo peptides in similar manner in the active center but cannot utilize the sulfur analogs as substrates. Instead, competitive inhibition occurs, which was further characterized for Ala-Gly-psi[CS-N]-Pro-Phe-NH-Np. The inhibition was nearly independent of the pH value in the range of pH 4.5-9, exhibiting apparent Ki values ranging from 200 to 600 microM. In comparison to Ala-Gly-trans-psi[CS-N]-Pro-Phe-NH-Np, the cis thioxo peptide Ala-Gly-cis-psi[CS-N]-Pro-Phe-NH-Np was found to possess an approximately 30-fold higher affinity for the active site of the enzyme. Thus, in the presence of stoichiometric amounts of Cyp18cy, the total amount of Ala-Leu-cis-psi[CS-N]-Pro-Phe-NH-Np in solution, detectable by isomer-specific proteolysis, was considerably enhanced.
Oligopeptides derived from the gag polyprotein (Pr55 gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55 gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cypl8). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag 21s-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-I Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The ICs0 value of 184 ~M for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1--4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPlase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cypl8, and may add a previously unrecognized topological component to the known subsite specificity of cyciophilins.
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