Pascal Demange

The structure of pyoverdine Pa, the siderophore of Pseudomonas aeruginosa

Abstract The structure of Pyoverdine Pa, the siderophore of Pseudomonas aeruginosa, was elucidated by degradation, by FAB mass spectrometry and by NMR; it consists of a linear octapeptide bound to a 2,3-diamino-6,7-dihydroxyquinoline derived chromophore.

Bacterial siderophores : structure and NMR assignment of pyoverdins Pa, siderophores ofPseudomonas aeruginosa ATCC 15692

SummaryIn iron-deficient conditions,Pseudomonas aeruginosa ATCC 15692 synthesizes two major siderophores, pyoverdins Pa and pyoverdin Pa B. Two other compounds, pyoverdin Pa A (occurring from hydrolysis of pyoverdin Pa during the culture) and pyoverdin Pa C (occurring artifactually during the purification procedure) were also isolated. All these compounds possess the same partly cyclic peptide chain wherel-Orn(δOH · HCO) isNδ-formyl,Nδ-hydroxy-l-ornithine. The chain is bound to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and having the (S) configuration. The four pyoverdins differ only in the acyl substituent bound to the nitrogen atom bound to carbon C3 of the chromophore. This is succinamide (pyoverdin Pa), succinic acid (pyoverdin Pa A), methyl succinate (pyoverdin Pa C) and 2-oxoglutaric acid (pyoverdin Pa B). The complete1H- and13CNMR assignments, using two-dimensional total correlation NMR spectroscopy (TOCSY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) procedures, as well as1H-13C correlations, are reported. The complete sequence of the peptide using CHα-NH correlations was achieved by NMR and confirmed the partly cyclic structure earlier reported using fast-atom-bombardment mass spectrometry (FAB-MS) on the siderophores and their dansylated fragments [Briskot G, Taraz K, Budzikiewicz H (1989)Liebigs Ann Chem: 375–384]. The use of these NMR procedures appears to be a tool of choice and a complementary approach to FAB-MS in the structure determination of some complex pyoverdins.

Structure-Function Analysis of the THAP Zinc Finger of THAP1, a Large C2CH DNA-binding Module Linked to Rb/E2F Pathways

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of ∼80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel β-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.

Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris

The human μ‐opioid receptor cDNA from which the 32 amino‐terminal codons were substituted by the Saccharomyces cerevisiae α‐mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase‐1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (K d = 0.2 nM and B max = 400 fmol/mg protein or 800 sites/cell). Competition studies with non‐selective, and μ‐, δ‐ and κ‐selective opioid agonists and antagonists revealed a typical μ‐opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.

Green fluorescent protein as a reporter of human μ-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris

Abstract The human μ-opioid receptor (HuMOR) was fused in its N-terminus end to the green fluorescent protein (GFP) or/and to the c-myc and six histidines tags in its C-terminus end, and expressed in the methylotrophic yeast Pichia pastoris. Neither the C- nor the N-terminal tagging of the receptor does modify its pharmacological properties as compared to the untagged receptor. Expression levels of fusion receptors determined by GFP fluorescence measurements strongly correlates with the number of sites expressed per cell detected through saturation studies (Bmax value), thus showing that GFP is an efficient and reliable reporter of the HuMOR functional expression. The N- and C-terminus tags have allowed to show that the entire molecule is overexpressed. They have permitted in-situ localization experiments using fluorescence and electron microscopy techniques and have shown a dense intracellular labelling. Above all, the quantification of expression levels made possible through fluorescence intensity analysis, have revealed that huge amounts of receptor are produced that could not be detected through classical binding experiments: for a Bmax value of 1 pmol mg−1 of receptor determined through binding studies, 16 pmol were found in membrane preparations using fluorescence and 100 pmol in whole cells. These results should be very useful for large-scale production and structural biology of HuMOR, and other G-protein coupled receptors (GPCRs).

Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts.

Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human μ-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%–90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water.

Bacterial siderophores: unusual 3,4,5,6-tetrahydropyrimidine-based amino acids in pyoverdins frompseudomonas fluorescens

Abstract Two unusual natural amino acids have been identified in the pyoverdins occurring from two different strains ofPseudomonas fluorescens. They contain a 3,4,5,6-tetrahydropyrimidine moiety resulting from the condensation of 2,4-diaminobutyric acid with the car☐yl group of respectively serine and glutamine.

The Transmembrane Protein KpOmpA Anchoring the Outer Membrane of Klebsiella pneumoniae Unfolds and Refolds in Response to Tensile Load

In Klebsiella pneumoniae the transmembrane β-barrel forming outer membrane protein KpOmpA mediates adhesion to a wide range of immune effector cells, thereby promoting respiratory tract and urinary infections. As major transmembrane protein OmpA stabilizes Gram-negative bacteria by anchoring their outer membrane to the peptidoglycan layer. Adhesion, osmotic pressure, hydrodynamic flow, and structural deformation apply mechanical stress to the bacterium. This stress can generate tensile load to the peptidoglycan-binding domain (PGBD) of KpOmpA. To investigate how KpOmpA reacts to mechanical stress, we applied a tensile load to the PGBD and observed a detailed unfolding pathway of the transmembrane β-barrel. Each step of the unfolding pathway extended the polypeptide connecting the bacterial outer membrane to the peptidoglycan layer and absorbed mechanical energy. After relieving the tensile load, KpOmpA reversibly refolded back into the membrane. These results suggest that bacteria may reversibly unfold transmembrane proteins in response to mechanical stress.

Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems IMPLICATIONS FOR THE ROLE OF THE NATURALLY OCCURRING MYCOLIC ACID MODIFICATION

PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. 15N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties.

Assignment of the configurations of the amino acids in peptidic siderophores

Pyoverdins and azotobactins contain beta-hydroxyaspartic acid, N delta-hydroxyornithine, citrulline and homoserine, in addition to the common protein amino acids. Configuration assignment of all of these was achieved by acid hydrolysis of the peptide, derivatization of the constituent amino acids to the N-pentafluoropropionyl amino acid esters and gas chromatographic separation of the stereoisomers on capillaries coated with Chirasil-Val. This approach is straightforward for the protein amino acids, but the less common amino acids are either partially degraded during acid hydrolysis or their derivatives exhibit unfavourable gas chromatographic properties. By judicious combination of partial and total hydrolysis and dual derivatization, these problems may be overcome.