Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.METASU

PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum): 1H﷓2-D NMR study and molecular modelling

International audiencePA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world. As such, PA1b is the first genuine protein phytotoxin specifically toxic to insects, which makes it a promising tool for seed weevil damage control. We have determined the 3-D solution structure of PA1b, using 2-D homonuclear proton NMR methods and molecular modeling. The primary sequence of the protein does not share similarities with other known toxins. It includes six cysteines forming three disulfide bridges. However, because of PA1b resistance to protease cleavage, conventional methods failed to establish the connectivity pattern. Our first attempts to assign the disulfide network from NOE data alone remained unsuccessful due to the tight packing of the cysteine residues within the core of the molecule. Yet, the use of ambiguous disulfide restraints within ARIA allowed us to establish that PA1b belongs to the inhibitor cystine-knot family. It exhibits the structural features that are characteristic of the knottin fold, namely, a triple-stranded antiparallel β-sheet with a long flexible loop connecting the first to the second strand and a series of turns. A comparison of the structural properties of PA1b with that of structurally related proteins adopting a knottin fold and exhibiting a diverse range of biological activities shows that the electrostatic and lipophilic potentials at the surface of PA1b are very close to those found for the spider toxin ACTX-Hi:OB4219, thereby suggesting activity on ion channels

Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogaster

The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms: they control heterotrimeric G-proteins, inhibit the MAP-kinase and NFκB signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure–function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism: Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes' expression and the corresponding proteins' functions, and to studying physiological mechanisms such as development. Here, we describe an optimized protocol for high level over-expression and high yield/high purity production of CG18594, one of Drosophila six putative PEBPs, for biophysical studies. The yield of the purified 15N labeled protein is estimated to be 60 mg/L of M9 minimal medium. Analysis of the secondary structure using circular dichroism indicates that the protein comprises mainly β-sheets at pH 7. The good dispersion of the crosspeaks on the 1H–15N HSQC spectrum provides evidence of a proper folding of the purified protein, though its time evolution suggests a tendency to denature. Taken together, these data are consistent with the assumption that the CG18594 protein belongs to the PEPB family

Legume AG41 peptide: a promising bio-insecticide

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A folded and functional synthetic PA1b, an interlocked entomotoxic miniprotein

1-ACL (articles avec comité de lecture)PA1b (Pea Albumin 1, subunit b) is a hydrophobic, 37-amino acid niiniprotein isolated from pea seeds (Pivum sativum), crosslinked by three interlocked disulfide bridges, signature of the ICK (inhibitory cystine-knot) family. It acts as an entomotoxic factor against major insect pests in stored crops and vegetables, making it a promising bioinsecticide. Here we report an efficient and simple protocol for the production of large quantities of highly pure, biologically active synthetic PA1b. The features of PA1b oxidative refolding revealed the off-pathway products and competitive aggregation processes. The efficiency of the oxidative folding can be significantly improved by using hydrophobic alcoholic cosolvents and decreasing the temperature. The homogeneity of the synthetic oxidized PA1b was established by reversed-phase HPLC. The correct pairing of the three disulfide bridges, as well as the three-dimensional structure of synthetic PA1b was assessed by NMR. Synthetic PA1b binds to rnicrosomal proteins from Sitophilus oryzae with a Kd of 8 nM, a figure quite similar to that determined for PA1b extracted from its natural source. Moreover, the synthetic miniprotein was as potent as the extracted one towards the sensitive strains of weevils. Our findings will open the way to the production of PA1b analogues by chemical means to an in-depth understanding of the PA1b mechanism of action