No abstract

Polypeptides often display proline-mediated conformational substates that are prone to isomer-specific recognition and function. Both possibilities can be of biological significance. Distinct families of peptidyl prolyl cis/trans isomerases (PPIases) evolved proved to be highly specific for proline moieties arranged in a special context of subsites. Structural and chemical features of molecules specifically bound to the active site of PPIases served to improve catalysis of prolyl isomerization rather than ground state binding. For example, results inferred from receptor Ser/Thr or Tyr phosphorylation in the presence of site-directed FKBP12 mutant proteins provided evidence for the crucial role of the enzymatic activity in downregulating function of FKBP12.z 1998 Federation of European Biochemical Societies.

Prolyl isomerases are folding enzymes and thus have the potential to catalyze their own folding. We show here that the folding of cytosolic FKBP12 (FK 506 binding protein) is an autocatalytic process both for the mature protein and for a fusion protein with an aminoterminal extension of 16 residues. Native FKBP contains seven trans-prolyl peptide bonds, and the cis-to-trans isomerizations of some or all of them constitute the slow, rate-limiting events in folding. The rate of an autocatalytic reaction increases with reactant concentration, because the product catalyzes its own formation. Accordingly, the folding of the fusion protein was more than 10-fold accelerated when the protein concentration was increased from 0.05 M to 10 M. At high concentrations of both forms of FKBP12 autocatalysis was very efficient, and the observed folding rate seemed to approach the rate of the fast direct folding reaction of the protein molecules with the correct (all trans) peptidyl-prolyl bond conformation.Protein folding in vitro and in vivo is often decelerated because it is coupled with prolyl isomerization (1-5). Cis/trans equilibria are usually established at the prolyl peptide bonds in an unfolded polypeptide chain, and, as a consequence, denatured proteins are heterogeneous mixtures of species which refold with different rates. Only the molecules with the nativelike prolines 1 can refold directly in a rapid reaction. The molecules with incorrect prolines refold in slow reactions that are limited in rate by the cis/trans isomerizations of these prolines. Proline-limited folding reactions are catalyzed in vitro and in vivo by peptidyl-prolyl cis/trans isomerases (3, 4, 6 -12). Four families of these ubiquitous proteins are now known (13-16), and their functions are not confined to catalyzing slow steps in protein folding (13,17 (24) and refolding can be followed by a change in protein fluorescence and by the regain of the prolyl isomerase activity.In an autocatalytic reaction the product accelerates its own formation and thus its rate is expected to increase with reactant concentration. For a proline-limited folding reaction, autocatalysis should thus lead to an increase in rate with protein concentration until the rate of direct fast refolding is approached. Therefore, in our refolding experiments, we varied the concentration of FKBP12 in a wide range, and in some folding experiments FK 506 was added to inhibit the prolyl isomerase activity of the refolded molecules. We found that autocatalysis contributes indeed to the refolding of most unfolded FKBP12 molecules. A strong autocatalytic rate enhancement was found in the folding of a variant of FKBP12 for which the direct and the proline-limited folding reactions differ vastly in rate. EXPERIMENTAL PROCEDURESMaterials-Urea (ultrapure) and guanidinium chloride (ultrapure) were from ICN Biomedicals, ␣-chymotrypsin and recombinant human cytoplasmic Cyp18 were from Boehringer-Mannheim, LiCl and CF 3 CH 2 OH were from Fluka, the assay peptide Suc-Ala-Phe-Pro-Phe-4-nitroanilide w...

Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.