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G Lippens

Publications and source records attributed to G Lippens.

At least 19 recordsLinked to original sources
PubMed2006

Exploring the molecular function of PIN1 by nuclear magnetic resonance.

PIN1 participates in the regulation of a number of signalling pathways in the cell involving protein phosphorylation/dephosphorylation. Its role seems to be an essential control level in addition to the protein phosphorylation by proline-directed kinases. Its cellular function includes regulation of the cell cycle by interaction with phosphorylated mitotic proteins such as Cdc25 and transcription factors such as p53. PIN1 was shown to be involved in the malignant transformation of cells in breast cancer, by up regulation of cyclinD1 and is thought to be involved in the development of the AD by regulating the function of phosphorylated Tau. We propose here to discuss the molecular function of PIN1 at the atomic level based on data from the recent literature and our own results obtained by the technique of Nuclear Magnetic Resonance. PIN1 specifically interacts with pThr/pSer-Pro motifs and is constituted by two domains: a WW N-terminal domain that binds pThr/pSer-Pro epitopes and a prolyl cis/trans isomerase C-terminal catalytic domain. An exception to this organisation is found in the plant PIN1 homologous enzymes, like PIN1At from Arabidopsis thaliana, that are constituted of the sole catalytic domain. The molecular function of PIN1, binding to and isomerization of pThr/pSer-Pro bonds, are thought to lead to several functional consequences. In a first mode of action, exemplified by its competition with the CKS protein, the interaction with PIN1 prevents interaction with other regulatory proteins, like ubiquitin-ligases that lead to degradation pathways. In a second mode of action, the idea is largely accepted that the local isomerization modifies the global conformation of the protein substrate and hence its intrinsic activity, although this has never been directly demonstrated. Finally, isomerization catalysis is thought to regulate the (de)phosphorylation of specific pThr/pSer-Pro motifs, exemplified by the stimulation of the dephosphorylation of pThr231 of Tau by the PP2A phosphatase.

Amino Acid Sequence↗
PubMed2001

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.

Amino Acid Sequence↗
PubMed2001

p13(SUC1) and the WW domain of PIN1 bind to the same phosphothreonine-proline epitope.

The WW domain of the human PIN1 and p13(SUC1), a subunit of the cyclin-dependent kinase complex, were previously shown to be involved in the regulation of the cyclin-dependent kinase complex activity at the entry into mitosis, by an unresolved molecular mechanism. We report here experimental evidence for the direct interaction of p13(SUC1) with a model CDC25 peptide, dependent on the phosphorylation state of its threonine. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)Calpha resonances during NMR titration experiments allows accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9(CKSHs2) by a sulfate molecule. The epitope recognized by p13(SUC1) includes the proline at position +1 of the phosphothreonine, as was shown by the decrease in affinity for a mutated CDC25 phosphopeptide, containing an alanine/proline substitution. No direct interaction between the PIN1 WW domain or its catalytic proline cis/trans-isomerase domain and p13(SUC1) was detected, but our study showed that in vitro the WW domain of the human PIN1 antagonizes the binding of the p13(SUC1) to the CDC25 phosphopeptide, by binding to the same phosphoepitope. We thus propose that the full cyclin-dependent kinase complex stimulates the phosphorylation of CDC25 through binding of its p13(SUC1) module to the phosphoepitope of the substrate and that the reported WW antagonism of p13(SUC1)-stimulated CDC25 phosphorylation is caused by competitive binding of both protein modules to the same phosphoepitope.

Amino Acid Sequence↗
PubMed2001

In vivo detection of the cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum by high-resolution magic angle spinning NMR.

We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

Glucans↗
PubMed2001

Rotor synchronization of radiofrequency and gradient pulses in high-resolution magic angle spinning NMR.

We have investigated the extent to which rotor synchronization of radiofrequency pulses leads to spectral improvement in high-resolution magic angle spinning NMR experiments. Several pulse sequences were tested, and the effect was found to be maximal in homonuclear TOCSY spectra. The physicochemical nature of the sample plays a role in the phenomenon, as rotor synchronization allows the refocusing of residual anisotropic interactions. However, even in a liquid sample the effects were visible. Radial inhomogeneities of the radiofrequency field were identified as an important source of the problem.

Magnetic Resonance Spectroscopy↗
PubMed2001

Solution structure of the main alpha-amylase inhibitor from amaranth seeds.

The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site.

Amaranth Dye↗
PubMed2001

High resolution magic angle spinning NMR in combinatorial chemistry.

Solid phase organic chemistry coupled with combinatorial methods promises to increase dramatically the diversity and number of small molecules available for medical and biological applications. However, optimizing the reaction conditions can be a time consuming step, especially since analytical tools to monitor reaction progress and detect impurities for solid phase chemistry are less developed than for solution chemistry. The use of high resolution magic angle spinning (HRMAS) NMR is described here as such an analytical tool. Whereas initial applications of molecular identification using deuterated organic solvents to swell the resins presented a significant gain in time over the cleave-and-analysis methods, the introduction of a differential diffusion filter has made immediate recording of spectra possible without any sample treatment. The applications of HRMAS NMR to different solid supports that are used in combinatorial chemistry will be described in terms of rapidity, robustness and sensitivity.

Chemistry, Organic↗
PubMed2000

Quantitative monitoring of solid phase organic reactions by high-resolution magic angle spinning NMR spectroscopy

Three possible high-resolution magic angle spinning (HR MAS) NMR experiments to quantitatively monitor a solid phase supported Horner-Emmons reaction are presented. In the first experiment we follow the solid phase reaction in deuterated solvent directly in the NMR rotor. The second quantification is done by reconditioning of a few milligrams of resin from an undefined reaction vessel by washing, drying, and reswelling in deuterated solvent, and the evaluation of the amount of resin bound structures by comparing to an external standard. The third experiment represents the first analytical quantification of resin-bound structures without any sample preparation, except the transfer of resin-solvent suspension (large excess of reagents in protonated dimethylformamide) from the reaction vessel to the NMR rotor.

Journal Article↗
PubMed2000

The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase.

A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana. The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1At mRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions.

Amino Acid Sequence↗
PubMed2000

MAS as a tool for suppressing dipolar field effects in high-resolution liquid NMR

We propose high-resolution magic angle spinning as a radical method for the suppression of dipolar field effects in liquid NMR. This technique works for any sample shape and any spatial distribution of nuclear magnetization. Furthermore, it removes any possibility of spectral clustering or instability due to the dipolar field. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

The Chemical Shift Index method applied to resin-bound peptides.

The Chemical Shift Index (CSI) method proposed by Wishart et al. [Biochemistry (1992) 31, 1647-1651] to evaluate the secondary structure of peptides in aqueous solution uses as its reference the chemical shift values of each of the 20 natural amino acids (X) in a typical nonstructured sequence GGXAGG (17-20). In order to apply the CSI method to protected resin-bound peptides, we established a new database of chemical shift values for the same GGXAGG sequences in their protected form and anchored to a polystyrene resin swollen in DMF-d7. The predictive value of this new reference set in the CSI protocol was tested on different resin-bound peptides that were previously characterized by a full NOE analysis.

Amino Acid Sequence↗
PubMed1999

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.

Antigens, CD↗
PubMed1999

Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.

The CKS1At gene product, p10CKS1At from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At, a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21(DE3) cotransformed with the CKS1At gene and the E. coli argU gene that encoded the arginine acceptor tRNAUCU produced a sufficient amount of p10CKS1At to start the structural study by NMR. Replacement of four rare codons in the CKS1At gene sequence, including a tandem arginine, by highly used codons in E. coli, restored also a high expression of the recombinant protein. Double-isotopic enrichment by 13C and 15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1At was purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1At protein.

Amino Acid Sequence↗
PubMed1999

Capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus. Structural study of an undecasaccharide obtained by lithium degradation.

The capsular polysaccharide produced by the thermophilic cyanobacterium Mastigocladus laminosus has been subjected to a specific degradation with lithium in ethylenediamine. The released undecasaccharide attached to one unit of tetrahydroxycyclopentanecarboxylic acid has been characterized by a combination of 2D nuclear magnetic resonance spectroscopy, mass spectrometry, monosaccharidic composition and linkage analyses. From the overlap of the structure of this oligosaccharide with previously identified di-, tri- and pentasaccharides released by mild acid hydrolysis, the capsular polysaccharide was deduced to have a pentadecasaccharide repeating unit with the following structure:

Carbohydrate Sequence↗
PubMed1999

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G.

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.

Amino Acid Sequence↗
PubMed1997

Solution structure of R-elafin, a specific inhibitor of elastase.

The solution structure of r-elafin, a specific elastase inhibitor, has been determined using NMR spectroscopy. Characterized by a flat core and a flexible N-terminal extremity, the three-dimensional structure is formed by a central twisted beta-hairpin accompanied by two external segments linked by the proteinase binding loop. A cluster of three disulfide bridges connects the external segments to the central beta-sheet and a single fourth disulfide bridge links the binding loop to the central beta-turn. The same spatial distribution of disulfide bridges can be observed in both domains of the secretory leukocyte protease inhibitor (SLPI), another elastase inhibitor. The structural homology between r-elafin and the C-terminal domain of SLPI confirms the former as a second member of the chelonianin family of proteinase inhibitors. Based on the homology between the two proteins and recent results obtained for elastase binding mutants of the bovine pancreatic trypsin inhibitor (BPTI), we define the segment 22 to 27 as the binding loop of elafin, with the scissile peptide bond between Ala24 and Met25. In our solution structures, this loop is extended and solvent-exposed, and exhibits a large degree of flexibility. This mobility, already observed for the binding loop in other protease inhibitors in solution, might be an important feature for the interaction with the corresponding protease.

Amino Acid Sequence↗
PubMed1997

Diagnosis of a case of acute chloroquine poisoning using 1H NMR spectroscopy: characterisation of drug metabolites in urine.

Analysis of biological fluids by proton nuclear magnetic resonance spectroscopy (1H NMR) is a promising tool in clinical biology. We have used this method for a rapid toxicological screening in the case of a suicide attempt. A urine sample was analysed at 300 and 600 MHz by 1D and 2D sequences (J-resolved and TOCSY) in a short experimental time. Quantification was realized by peak integration of the 1D spectrum. The results showed the presence of chloroquine and its major metabolite monodesethylchloroquine at concentrations of 462 and 140 mg/L, respectively. Ethanol was also detected in the spectrum. It can be concluded that 1H NMR provides many advantages as a tool for clinical diagnosis in a case of acute intoxication.

Acute Disease↗