Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca²⁺-loaded calmodulin (Ca²⁺/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca²⁺/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca²⁺/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with ¹⁵N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca²⁺-dependent manner with the Par17 N terminus. The reverse experiment with ¹⁵N-labeled Ca²⁺/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca²⁺/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK^796–815 complex. In vitro tubulin polymerization assays furthermore showed that Ca²⁺/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca²⁺/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca²⁺/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca²⁺ signaling with microtubule function.     

Structure and dynamics of the first archaeal parvulin reveal a new functionally important loop in parvulin-type prolyl isomerases

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the β-α₃-β-α-β₂ fold typical for all parvulin structures known so far, but with helix III being a short 3₁₀-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 3₁₀-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for ¹H-¹⁵N-HSQC titrations. Again, the flexible region around 3₁₀-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.     

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

Background  

The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study.     

Parvulin (Par14), a Peptidyl-Prolyl cis-trans Isomerase, Is a Novel rRNA Processing Factor That Evolved in the Metazoan Lineage

Although parvulin (Par14/eukaryotic parvulin homolog), a peptidyl-prolyl cis-trans isomerase, is found associated with the preribosomal ribonucleoprotein (pre-rRNP) complexes, its roles in ribosome biogenesis remain undetermined. In this study, we describe a comprehensive proteomics analysis of the Par14-associated pre-rRNP complexes using LC-MS/MS and a knockdown analysis of Par14. Together with our previous results, we finally identified 115 protein components of the complexes, including 39 ribosomal proteins and 54 potential trans-acting factors whose yeast homologs are found in the pre-rRNP complexes formed at various stages of ribosome biogenesis. We give evidence that, although Par14 exists in both the phosphorylated and unphosphorylated forms in the cell, only the latter form is associated with the pre-40 S and pre-60 S ribosomal complexes. We also show that Par14 co-localizes with the nucleolar protein B23 during the interphase and in the spindle apparatus during mitosis and that actinomycin D treatment results in the exclusion of Par14 from the nucleolus. Finally we demonstrate that knockdown of Par14 mRNA decelerates the processing of pre-rRNA to 18 and 28 S rRNAs. We propose that Par14 is a component of the pre-rRNA complexes and functions as an rRNA processing factor in ribosome biogenesis. As the amino acid sequence of Par14 including that in the amino-terminal pre-rRNP binding region is conserved only in metazoan homologs, we suggest that its roles in ribosome biogenesis have evolved in the metazoan lineage.Peptidyl-prolyl cis-trans isomerases (PPIases)¹ catalyze the rotation about the peptide bond on the amino-terminal side of proline, a step that can be rate-limiting for the folding of newly synthesized proteins (1). PPIases also have the ability to bind many proteins, thereby acting as chaperones; thus, they are believed to control the activity of proteins by regulating their folding, assembly, and intracellular trafficking (2–4). There are three families of PPIases, namely the cyclophilin (CyP), FK506-binding protein, and parvulin families. The CyP and FK506-binding protein families have been well established as targets of the immunosuppressants cyclosporin A and FK506, respectively (5–7).Together with Pin1, human parvulin (Par14, EPVH) constitutes the parvulin family and has been identified in all hitherto examined human tissues (8, 9). Par14 comprises 131 amino acid residues and has a 35-residue amino-terminal region that does not have sequence similarity to the WW domain (known to bind to phosphorylated serine/threonine-proline bonds in proteins and peptides) of Pin1. Phosphorylation at Ser-19 in this region regulates the subcellular localization and DNA binding activity of Par14; the phosphorylation is required for nuclear localization, and the dephosphorylation is a prerequisite for the binding of the first 25 residues to nuclear DNA (10). The 96-residue carboxyl-terminal domain has a 34.2% sequence identity with the PPIase domain of Pin1. Par14 reportedly has a substrate preference for positively charged residues preceding proline but not for phosphorylated Thr or Ser as is the case with Pin1; however, its rate constant for the prolyl cis to trans isomerization reaction is at least 1,000-fold lower than that of CyPs (9). NMR solution structural analysis has shown that Par14 folds into a βα3βαβ2 structure, which is essentially identical to that of Pin1 (11). The unstructured 35-residue amino-terminal region contains several basic residues and replaces the WW domain of Pin1 (11). This structural model explains the molecular basis for the preferential substrate specificity of Par14 for positively charged residues preceding proline as well as the putative role of the amino-terminal region as a DNA-binding domain. However, the physiological function of Par14 remains unknown.We previously reported that Par14 associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes as well as with many proteins that are implicated in the regulation of microtubule assembly or nucleolar reformation during mitosis (12, 13). We have proposed that Par14 is involved in ribosome biogenesis and/or nucleolar reassembly in mammalian cells during the pre- or postmitotic phases of the cell cycle. In the present study, we describe the comprehensive identification of protein components of the Par14-associated pre-rRNP complexes and establish Par14 as a de facto component of the pre-rRNP complexes in vivo. We also demonstrate that Par14 functions as a ribosomal RNA processing factor in mammalian ribosome biogenesis.     

Structure and plasticity of the peptidyl-prolyl isomerase Par27 of Bordetella pertussis revealed by X-ray diffraction and small-angle X-ray scattering

Par27 from Bordetella pertussis belongs to a newly discovered class of dimeric peptidyl-prolyl isomerase (PPIase)/chaperones from the parvulin family. It is a tripartite protein with a central PPIase domain surrounded by N- and C-terminal sub-domains (NTD and CTD). Here, the Par27 structure was characterized by X-ray crystallography, small-angle X-ray scattering and template-based modeling. In the crystal lattice, Par27 consists of alternating well ordered and poorly ordered domains. The PPIase domains gave rise to diffuse scattering and could not be solved, whereas a 2.2 Å resolution crystal structure was obtained for the NTD and CTD, revealing a cradle-shaped dimeric platform. Despite a lack of sequence similarity with corresponding sub-domains, the topology of the peptide chain in the NTD/CTD core is similar to that of other monomeric PPIase/chaperones such as SurA and trigger factor from Escherichia coli. In Par27, dimerization occurs by sub-domain swapping. Because of the strong amino acid sequence similarity to other parvulin domains, a model for the Par27 PPIase domain was built by template-based modeling and validated against small-angle X-ray scattering (SAXS) data. A model of the full-length dimeric Par27 structure was built by rigid-body modeling and filtering against SAXS data using the partial crystal structure of the NTD/CTD core and the template-based PPIase model. The flexibility of protein was accounted for by representing the structure as an ensemble of different conformations that collectively reproduce the scattering data. The refined models exhibit a cradle-like shape reminiscent of other PPIase/chaperones, and the variability in the orientation of the PPIase domains relative to the NTD/CTD core platform observed in the different models suggests inter-domain flexibility that could be important for the biological activity of this protein.     

Quantum mechanical calculations and experimental measurement of N-terminal charge effects on 1HN and 1HCα chemical shifts in peptides

Nuclear magnetic resonance spectroscopists are increasingly utilizing chemical shifts to characterize the secondary structure of proteins. The present study addresses the effects that the positively charged amino group at the N-terminus of a peptide has on ¹HN and ¹HCα chemical shifts along the chain. This information is necessary for interpreting chemical shift data for proteins and/or for peptides that are used as models for protein structure. The chemical shifts for the ¹H resonances of four peptides that differ only in the location of their N-terminii are assigned using two-dimensional nmr spectroscopy. The peptides have sequences derived from the β subunit of the glycoprotein hormone human chorionic gonadotropin (hCG-β). Comparison of the ¹HN and the ¹HCα chemical shifts for residues common to all four peptides reveals downfield shifts for ¹HN and the ¹HCα resonances within three residues of the N-terminus compared with chemical shifts in the interior of the peptide. The magnitude of the downfield shift is larger for resonances nearer the N-terminus. Quantum mechanical calculations of the ¹HN and ¹HCα chemical shifts in peptides constructed with six alanine units also predict a significant terminus effect. The calculations agree both qualitatively and quantitatively with the experimental data. The inductive nature of the end effect is confirmed in the calculations by Mulliken population analysis. End effects should be taken into account in determining protein secondary structures from chemical shifts. © 1996 John Wiley & Sons, Inc.     

The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N‐terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin‐like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two‐state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease‐free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline‐containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.

     

The peptidyl-prolyl isomerase and chaperone Par27 of Bordetella pertussis as the prototype for a new group of parvulins

Proteins that pass through the periplasm in an unfolded state are highly sensitive to proteolysis and aggregation and, therefore, often require protection by chaperone-like proteins. The periplasm of Gram-negative bacteria is well equipped with ATP-independent chaperones and folding catalysts, including peptidyl-prolyl isomerases (PPIases). The filamentous hemagglutinin of Bordetella pertussis, which is secreted by the two-partner secretion pathway, crosses the periplasm in an unfolded conformation. By affinity chromatography, we identified a new periplasmic PPIase of the parvulin family, Par27, which binds to an unfolded filamentous hemagglutinin fragment. Par27 differs from previously characterized bacterial and eukaryotic parvulins. Its central parvulin-like domain is flanked by atypical N- and C-terminal extensions that are found in a number of putative PPIases present mostly in β proteobacteria. Par27 displays both PPIase and chaperone activities in vitro. In vivo, Par27 might function as a general periplasmic chaperone in B. pertussis.     

Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The C^αH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected C^βH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

Evaluating amber force fields using computed NMR chemical shifts

NMR chemical shifts can be computed from molecular dynamics (MD) simulations using a template matching approach and a library of conformers containing chemical shifts generated from ab initio quantum calculations. This approach has potential utility for evaluating the force fields that underlie these simulations. Imperfections in force fields generate flawed atomic coordinates. Chemical shifts obtained from flawed coordinates have errors that can be traced back to these imperfections. We use this approach to evaluate a series of AMBER force fields that have been refined over the course of two decades (ff94, ff96, ff99SB, ff14SB, ff14ipq, and ff15ipq). For each force field a series of MD simulations are carried out for eight model proteins. The calculated chemical shifts for the ¹H, ¹⁵N, and ¹³C^a atoms are compared with experimental values. Initial evaluations are based on root mean squared (RMS) errors at the protein level. These results are further refined based on secondary structure and the types of atoms involved in nonbonded interactions. The best chemical shift for identifying force field differences is the shift associated with peptide protons. Examination of the model proteins on a residue by residue basis reveals that force field performance is highly dependent on residue position. Examination of the time course of nonbonded interactions at these sites provides explanations for chemical shift differences at the atomic coordinate level. Results show that the newer ff14ipq and ff15ipq force fields developed with the implicitly polarized charge method perform better than the older force fields.     

Chemical shift-dependent apparent scalar couplings: an alternative concept of chemical shift monitoring in multi-dimensional NMR experiments

The paper presents an alternative technique for chemical shift monitoring in a multi-dimensional NMR experiment. The monitored chemical shift is coded in the line-shape of a cross-peak through an apparent residual scalar coupling active during an established evolution period or acquisition. The size of the apparent scalar coupling is manipulated with an off-resonance radio-frequency pulse in order to correlate the size of the coupling with the position of the additional chemical shift. The strength of this concept is that chemical shift information is added without an additional evolution period and accompanying polarization transfer periods. This concept was incorporated into the three-dimensional triple-resonance experiment HNCA, adding the information of ¹H chemical shifts. The experiment is called HNCA^codedHA, since the chemical shift of ¹H is coded in the line-shape of the cross-peak along the ¹³C dimension.     

13C-nmr chemical shift and conformation of L-alanine residues incorporated into poly(β-benzyl L-aspartate) in the solid state

¹³C-nmr spectra of poly(β-benzyl L-aspartate) containing ¹³C-enriched [3-¹³C]L -alanine residues in the solid state were recorded by the cross polarization–magic angle spinning method, in order to elucidate the conformation-dependent ¹³C chemical shifts of L -alanine residues taking various conformations such as the antiparallel β-sheet, the right-handed α-helix, the left-handed α-helix, and the left-handed ω-helix forms obtained by appropriate treatment. The latter two conformations for L -alanine residues are achieved when L -alanine residues are incorporated into poly(β-benzyl L -aspartate). We found that the alanine C_β carbon show significant ¹³C chemical shift displacement depending on conformational change, and gave the ¹³C chemical shift values at about 17 ppm for the left-handed ω-helix, 14 ppm for the left-handed α-helix, 15.5 ppm for the right-handed α-helix, and 21.0 ppm for the antiparallel β-sheet relative to tetramethylsilane.     

Identification and characterization of peptides that bind the PPIase domain of Parvulin17

Peptidyl‐prolyl cis‐trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N‐terminal to the proline substrate. Par14 and its isoform Par17 belong to the Parvulin family of PPIases. Par14 can bind AT‐rich double‐stranded DNA and was shown to be part of the pre‐ribosomal ribonucleoprotein (pre‐rRNP) complexes, where it functions as an RNA processing factor that is involved in ribosome biogenesis. Its longer isoform Par17 is expressed only in cells of hominids, where it is targeted to the mitochondria. To find binding partners (peptides or proteins) for Par17, we applied the phage display technology. We panned 7‐mer and 12‐mer peptide libraries against Par17. The consensus sequence XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid, was enriched from both libraries. We demonstrate the binding of this motif to the PPIase domain of Par17 using phage ELISA and NMR spectroscopy. We propose that residues Met90, Val91, Phe94, Gln95, Glu96, and Ala98 of Par17 are involved in substrate recognition, and that the phage display‐selected motif XHSXVHØ can be recognized by Par17 PPIase domain in vivo. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Automatic <Superscript>13</Superscript>C chemical shift reference correction for unassigned protein NMR spectra

Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR, including protein structure determination and analysis of protein dynamics. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct ¹³C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ±?0.4 ppm of the correct ¹³C reference value. On a much larger unassigned chemical shift test set, the base method kept ¹³C chemical shift referencing errors to within ±?0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct ¹³C chemical shift referencing errors to within ±?0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct ¹³C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. These new methods will allow non-NMR experts to detect and correct ¹³C referencing error at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis.     

Pressure effect on the dynamics of an isolated α-helix studied by 15N-1H NMR relaxation

Dynamics and structure of (1–36)bacteriorhodopsin solubilized in chloroform/methanol mixture (1:1) were investigated by ¹H-¹⁵N NMR spectroscopy under a hydrostatic pressure of 2000 bar. It was shown that the peptide retains its spatial structure at high pressure. ¹⁵N transverse and longitudinal relaxation times, ¹⁵N{¹H} nuclear Overhauser effects, chemical shifts and the translation diffusion rate of the peptide at 2000 bar were compared with the respective data at ambient pressure [Orekhov et al. (1999) J. Biomol. NMR, 14, 345–356]. The model free analysis of the relaxation data for the helical 9–31 fragment revealed that the high pressure decreases the overall rotation and translation diffusion, as well as apparent order parameters of fast picosecond internal motions (S² _f) but has no effect on internal nanosecond motions (S² _s and _s) of the peptide. The decrease of translation and overall rotation diffusion was attributed to the increase in solvent viscosity and the decrease of apparent order parameters S² _f to a compression of hydrogen bonds. It is suggested that this compression causes an elongation of H-N bonds and a decrease of absolute values of chemical shift anisotropy (CSA). In particular, the observed decrease of S² _f at 2000 bar can be explained by 0.001 nm increase of N-H bond lengths and 10 ppm decrease of ¹⁵N CSA values.     

Generation of a Highly Active Folding Enzyme by Combining a Parvulin-Type Prolyl Isomerase from SurA with an Unrelated Chaperone Domain

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.     

Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, β-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of β-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules.