Binding characteristics of [3H]-JSM10292: a new cell membrane-permeant non-peptide bradykinin B2 receptor antagonist.

BACKGROUND AND PURPOSE: A (3) H-labelled derivative of the novel small-molecule bradykinin (BK) B(2) receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B(2) receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH: Equilibrium binding, dissociation and competition studies with various B(2) receptor ligands and [(3) H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B(2) receptors of human and animal origin. KEY RESULTS: [(3) H]-JSM10292 bound to B(2) receptors at 4°C and at 37°C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B(2) receptor agonists or antagonists were added. [(3) H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B(2) receptors, as indicated by the different 'nonspecific' binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [(3) H]-JSM10292 and competition experiments with unlabelled JSM10292 and [(3) H]-BK showed a different affinity profile for the wild-type B(2) receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B(2) receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B(2) receptor ligand. CONCLUSIONS AND IMPLICATIONS: [(3) H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B(2) receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B(2) receptors.

Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains.

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

Identification and characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase.

A second member of the parvulin family of peptidyl-prolyl cis/trans isomerases was identified in a human lung cDNA library. The gene encoded a protein named hPar14 that has 131 amino acid residues and a molecular mass of 13676 Da. Sequence comparison showed 34.5% identity to E. coli Par10 and 34% identity to human Pin1 (hPar18) within a C-terminal region of 87 or 120 amino acid residues, respectively. In comparison to the E. coli Par10, hPar14 possesses a N-terminal extension of 41 amino acid residues. This extension does not contain a polyproline II helix-binding motif typical of the known eukaryotic parvulins. The hPar14 does not accelerate the cis to trans interconversion of oligopeptides with side chain-phosphorylated Ser(Thr)-Pro moieties as hPin1 did. In contrast, it showed preference of an arginine residue adjacent N-terminal to proline. Northern blot analysis revealed expression of the gene within various human tissues like heart, placenta, liver, kidney and pancreas.

Effects of FK506-binding protein 12 and FK506 on autophosphorylation of epidermal growth factor receptor.

FK506-binding proteins and cyclophilins are intracellular proteins that express peptidylproline cis-trans-isomerase (PPIase) activity. The effects of FK506-binding protein 12 (FKBP12) and the cyclophilins 18 and 23 on autophosphorylation of the epidermal growth factor (EGF) receptor prepared from plasma membranes of the human epidermoid cell line A431 have been investigated. Whereas FKBP12 inhibited EGF receptor tyrosine kinase activity in a concentration-dependent manner, the cyclophilins did not affect autophosphorylation. In contrast to the wild-type enzyme, several variants of FKBP12 with greatly reduced PPIase activity were unable to suppress EGF receptor tyrosine kinase significantly. Pervanadate an inhibitor of protein tyrosine phosphatases, abolished the effect of FKBP12 on EGF receptor autophosphorylation. Finally, FK506 and rapamycin, which are known to block the PPIase activity of FKBP12, induced a significant stimulation of EGF receptor autophosphorylation in intact A431 cells suggesting suppression of EGF receptor autophosphorylation by intracellular FKBP12 in vivo. Taken together the data point to an inhibitory function of FKBP12 in EGF receptor signaling, possibly induced by stimulation of a protein tyrosine phosphatase coupled to the EGF receptor. Both PPIase activity and substrate specificity of FKBP12 seem to be indispensable for this effect.

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.

Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12.

A low degree of amino acid sequence similarity to FK506-binding proteins (FKBPs) has been obtained for the peptidyl prolyl cis/trans isomerase (PPIase) domain of E. coli trigger factor (TF) that was thought to be significant with regard to the enzymatic properties of the bacterial enzyme. We examined whether the alteration of a negatively charged side-chain at position 37 (FKBP numbering) and a phenylalanine at position 99, both highly conserved through both types of enzymes, leads to parallel effects on the catalytic activity of both FKBP12 and TF-PPIase domain in a series of tetrapeptide substrates with different P1 subsites. For the latter enzyme, substitution of Glu178 by Val or Lys, which aligns to Asp37 in human FKBP12, enhanced the PPIase activity, whereas a strongly decreased enzymatic activity was determined for the Asp37Leu and Asp37Val variants of FKBP12. Regardless of the P1 subsite of the substrate used for the assay, mutation of Phe233Tyr generated a protein variant of the TF-PPIase domain with about 1% of the wild type PPIase activity. Dependent on the substrate nature, a moderate decrease as well as a 4.8-fold increase in k(cat)/K(M) could be determined for the corresponding Phe99Tyr FKBP12 variant. Neither of the mutations of the TF-PPIase domain was able to implant FK506 inhibition found as a major characteristic of the FKBP family of PPIases.

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila--characterization, molecular cloning and overexpression.

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires' disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPlase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPlases. The Icy gene (Legionella cyclophilin) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17968 Da called L. pneumophila cyclophilin 18 (L.p.Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coli with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histidine tag and an enterokinase cleavage site exhibits PPlase activity when produced at high levels in E. coli K-12. After removal of the extension by enterokinase, the properties of the recombinant Cyp18 were indistinguishable from those of the authentic enzyme. In order to investigate the influence of Cyp18 on intracellular survival of L. pneumophila an Icy-negative L. pneumophila strain was constructed. Compared with the wild-type strain, the mutant did not exhibit a significant phenotype but was 10-fold less invasive for Acanthamoeba castellanii. Like human cyclophilin, the L. p. Cyp18 exhibits nuclease activity, but this enzymatic activity does not appear to be linked with the native structure of the protein.

An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity.

The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cis/trans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatically active fragment could be isolated after proteolysis by subtilisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni2+-NTA column. Subsequent digestion with subtilisin and anion-exchange chromatography yielded a TF fragment encompassing amino acids Gln-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 microM(-1) s(-1) could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrolide FK506.