Increased backbone flexibility in threonine45-phosphorylated hirudin upon pH change.

Protein phosphorylation on serine/threonine side chains represents a major regulatory event in the posttranslational control of protein functionality, where it is thought to operate at the level of structural changes in the polypeptide chain. However, key questions about molecular aspects of phosphate ester induced conformational alterations remain open. Among these concerns are the radius of action of the phosphate ester group, its effective ionic state, and its interplay with distinct bonds of the polypeptide chain. Primarily to define short-range effects upon threonine phosphorylation, the native 65 amino acid protein hirudin, conformationally restrained by a proline flanking the pThr(45) site and three intramolecular disulfide bonds, was structurally characterized in both the phosphorylated and the unphosphorylated state in solution. Circular dichroism and hydrogen exchange experiments (MALDI-TOF) showed that structural changes were caused by Thr(45)-Pro(46) phosphorylation only when the phosphate ester group was in its dianionic state. The spatial arrangement of the amino acids, monitored by 1H NMR spectroscopy, appears to be affected within a radius of about 10 A around the pThr(45)-OgammaH, with phosphorylation resulting in a loss of structure and increased flexibility within a segment of at least seven amino acid residues. Thus, the transition from the monoanionic to the dianionic phosphate group over the pH range 5.2-8.5 represents a general phosphorylation-dependent conformational switch operating at physiological pH values.

The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis.

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.

Modular structure of the trigger factor required for high activity in protein folding.

The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans isomerase (PPIase) which catalyzes proline-limited protein folding extremely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments which encompass individual domains or combinations of the middle FKBP-like domain (M) with the N-terminal (N) and C-terminal (C) regions, respectively. These fragments appear to be stably folded. They show ordered structure and cooperative urea-induced unfolding transitions, and the far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the native trigger factor shows a modular structure, which is composed of three fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activity in protein folding could not be restored by fusing alternatively the N or the C-terminal regions to the catalytic domain (in NM and MC constructs, respectively). Surprisingly, the high folding activity of the intact trigger factor has been regained partially by functional complementation of the overlapping NM and MC constructs.

Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding.

The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. In its enzymatic mechanism the trigger factor follows the Michaelis-Menten equation. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 microM. In contrast, the catalytic constant kcat is small and shows a value of 1.3 s(-1) at 15 degrees C. An unfolded protein inhibits the trigger factor in a competitive fashion. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. Together, this suggests that the good substrate binding, i.e. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding.

Autocatalytic folding of the folding catalyst FKBP12.

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 amino-terminal 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 microM to 10 microM. 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.

Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond.

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.