On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate

Modulation of RXR-DNA complex assembly by DNA context

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Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins: From Expression Screening for Soluble Constructs to Protein Quality Control

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Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of α-Helix As Oxyanion-Binding Site

Design of catalytic proteins with functional sites capable of specific chemistry gains its momentum and a number of artificial enzymes have been recently reported including hydrolases, oxidoreductases, retro-aldolases and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation and possessing no stringent restrictions to the amino acid composition at the ligation junction. Here we report the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied minimalist approach to rationally design an oxyanion hole within a small cavity and containing an adjacent thiol nucleophile. The N-terminus of alpha-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition-elimination reactions at acyl group. Cysteine acting as a principal catalytic residue was introduced at second residue position of alpha-helix N-terminus in a designed three-alpha-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide-thioesters as acyl-donors we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation and peptide ligation

Aminobenzosuberone derivatives as PfA-M1 inhibitors: Molecular recognition and antiplasmodial evaluation

International audienceAminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC50 values of 6.5-11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein

Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR–RXR

International audienceRetinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNAbinding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers

Sci Rep

Retinoid X receptors (RXRs) act as homodimers or heterodimerisation partners of class II nuclear receptors. RXR homo- and heterodimers bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1). We present a structural characterization of RXR-DNA binding domain (DBD) homodimers on several natural DR1s and an idealized symmetric DR1. Homodimers displayed asymmetric binding, with critical high-affinity interactions accounting for the 3' positioning of RXR in heterodimers on DR1s. Differing half-site and spacer DNA sequence induce changes in RXR-DBD homodimer conformation notably in the dimerization interface such that natural DR1s are bound with higher affinity than an idealized symmetric DR1. Subtle changes in the consensus DR1 DNA sequence therefore specify binding affinity through altered RXR-DBD-DNA contacts and changes in DBD conformation suggesting a general model whereby preferential half-site recognition determines polarity of heterodimer binding to response elements

TJ recruitment of TRAF4 is PIP-binding dependent.

<p>(A) Schematic representation of sh-insensitive Flag-tagged WT and mutant TRAF4 constructs used to reintroduce TRAF4 expression in MCF7/shT4 cells. (B) Recruitment of WT and mutant TRAF4 proteins (green) at TJs was analyzed by colocalization with ZO-1 (red). The highlighted overlap (white) between TRAF4 and ZO-1 staining is shown on merge panels and alone on the right panel. While the TRAF4-K313E mutant is still partially colocalized with ZO-1 (middle panels), the TRAF4-K345E mutant does not colocalize anymore with ZO-1 (bottom panels). Scale bar, 10 µm. (C) Quantification of WT and mutant TRAF4 recruitment at TJs. The colocalization index (overlapping area between TRAF4 and ZO-1 staining divided by the TJ length) was measured on 10 microscopic fields. Compared to the WT protein, the colocalization index was reduced by 40% and 78% for K313E and K345E TRAF4 mutants, respectively. (D) Western blot analysis of TRAF4 protein level in parental MCF10A and in TRAF4-silenced cells (MCF10A/shT4) where WT (MCF10A/shT4+TRAF4) and mutant (MCF10A/shT4+TRAF4K345E) TRAF4 expression was restored. The MCF10A/shT4+pBABE cell line represents a control line transduced with the empty vector. Beta-actin was used as a loading control. (E) The presence of TJs was estimated by ZO-1 staining in parental (a) and in TRAF4-silenced cells where the expression of WT (c) and mutant TRAF4 (d) was reintroduced. TRAF4-silenced cell line transduced with the empty vector (b) was used as a control. The PIP-binding–deficient TRAF4-K345E cannot rescue the phenotype induced by TRAF4 silencing on TJs. Left panels, representative confocal image sections of ZO-1 staining (green); right panels, merge with Hoechst staining (blue). Scale bar, 20 µm. (F) TJ quantification in cell lines described in (D) and (E) was performed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001726#pbio-1001726-g001" target="_blank">Figure 1E</a>. n, number of microscopic fields used for the quantification.</p

TRAF4 binds PIPs through its TRAF domain.

<p>(A) Schematic representation of the recombinant proteins used in lipid binding assays. TAP and 6His tags were used for the purification. RING, Zf, and TRAF are conserved structural domains present in the TRAF4 protein. (B) Coomassie blue staining (a) and Western blot analysis (b) of purified recombinant proteins. The antibody used recognized the immunoglobulin-binding domain of protein A from the TAP tag. TRAF4 degradation products are indicated by asterisks. (C) Lipid-overlay assay. Left, schematic view of a PIP-strip membrane. LPA, lysophosphatidic acid; S1P, Sphingosine-1-phosphate; LPC, Lysophosphocholine; PI, Phosphatidylinositol; PI(3)P, PI-(3)-phosphate; PI(4)P, PI-(4)-phosphate; PI(5)P, PI-(5)-phosphate; PI(3,4)P2, PI-(3,4-)bisphosphate; PI(3,5)P2, PI-(3,5)-bisphosphate; PI(4,5)P2, PI-(4,5)-bisphosphate; PIP(3,4,5)P3, PI-(3,4,5)-trisphosphate; PA, Phosphatidic acid; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PC, Phosphatidylcholine. The TAP-6His recombinant protein served as a negative control. Immunodetection of bound proteins was performed using a TAP-identifying antibody. TAP-6His and RING-7xZf did not bind to any membrane-coated lipids, while both full-length TRAF4 and the TRAF domain in isolation interacted with all PIPs and PA. (D) The TRAF domain of TRAF4 binds PIP in solution. Electrospray ionization time-of-flight mass spectrometry deconvoluted spectra of the TRAF domain of TRAF4 in the absence (a) and in the presence (b) of PI(3,4,5)P3-diC4. In isolation and in the absence of lipid, the TRAF domain is a trimer (a). In the presence of PIP, three additional peaks corresponding to one to three bound lipids are detected (b). Theoretical masses of the TRAF trimer and PI(3,4,5)P3-diC4 are 67.678 kDa and 0.714 kDa, respectively. (E) Liposomes flotation assay. a, schematic representation of the liposome flotation assay. Blank liposomes, PI(4,5)P2-containing liposomes, and PI(3,4,5)P3-containing liposomes were incubated with recombinant proteins, and liposome/protein-mixed fractions were separated by sucrose gradient ultracentrifugation. Binding of recombinant control TAP-6HIS (b) and TRAF domain of TRAF4 (c) to liposomes using membrane flotation assay. Fluorescent analyses (dot blot) of NBD-PE indicated that blank and PIP-containing liposomes were present in the top fraction. The presence of recombinant proteins in each fraction was detected by Western blot using anti-His antibody and quantified by densitometry-analysis using ImageJ software. The control TAP-6His was predominantly detected in the bottom fraction (b). In contrast the centrifugation profile of the TRAF4-TRAF domain was modified in the presence of PIP-containing liposomes (c). Indeed when mixed with blank liposomes, the TRAF domain was present in the bottom fraction, while in the presence of PI(4,5)P2) and PI(3,4,5)P3-containing liposomes, the TRAF domain was present in the top fraction. (F) Affinity of TRAF4 for PIP was measured by ITC. Titration was performed with 16 µM TRAF-6His recombinant protein, to which 500 µM of inositol-(1,3,4,5)-tetrakisphosphate were added incrementally. The TRAF domain of TRAF4 binds IP4 with a K_D of 5.68 µM.</p