Cédric Bernard

Recombinant production and solution structure of PcTx1, the specific peptide inhibitor of ASIC1a proton‐gated cation channels

Acid‐sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton‐sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a spider toxin. The recombinant toxin was identical in all respects to the native peptide, and its three‐dimensional structure in solution was determined by means of 1H 2D NMR spectroscopy. Surface characteristics of PcTx1 provide insights on key structural elements involved in the binding of PcTx1 to ASIC1a channels. They appear to be localized in the β‐sheet and the β‐turn linking the strands, as indicated by electrostatic anisotropy calculations, surface charge distribution, and the presence of residues known to be implicated in channel recognition by other inhibitor cystine knot (ICK) toxins.

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein

In this report, the solution structure of the nucleocapsid‐binding domain of the measles virus phosphoprotein (XD, aa 459–507) is described. A dynamic description of the interaction between XD and the disordered C‐terminal domain of the nucleocapsid protein, (NTAIL, aa 401–525), is also presented. XD is an all α protein consisting of a three‐helix bundle with an up‐down‐up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489–491, which is involved in the embedding of the α‐helical XD‐binding region of NTAIL. Secondary chemical shift values for full‐length NTAIL were used to define the precise boundaries of a transient helical segment that coincides with the XD‐binding domain, thus shedding light on the pre‐recognition state of NTAIL. Titration experiments with unlabeled XD showed that the transient α‐helical conformation of NTAIL is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483–506 of NTAIL are in intermediate exchange with XD, while the 475–482 and 507–525 regions are in fast exchange. The NTAIL resonance behavior in the titration experiments is consistent with a complex binding model with more than two states. Copyright

Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels.

Acid‐sensing ion channels (ASIC) are proton‐gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42‐amino‐acid peptide cross‐linked by three disulfide bridges. Its three‐dimensional structure, as determined by conventional two‐dimensional 1H‐NMR, consists of a compact disulfide‐bonded core composed of a four‐stranded β‐sheet. It belongs to the disulfide‐rich all‐β structural family encompassing peptide toxins commonly found in animal venoms. The structural characteristics of APETx2 are compared with that of PcTx1, another effector of ASIC channels but specific to the ASIC1a subtype and to APETx1, a toxin structurally related to APETx2, which targets the HERG potassium channel. Structural comparisons, coupled with the analysis of the electrostatic characteristics of these various ion channel effectors, led us to suggest a putative channel interaction surface for APETx2, encompassing its N terminus together with the type I‐β turn connecting β‐strands III and IV. This basic surface (R31 and R17) is also rich in aromatic residues (Y16, F15, Y32, and F33). An additional region made of the type II′‐β turn connecting β‐strands I and II could also play a role in the specificity observed for these different ion effectors.

Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus

Animal toxins block voltage‐dependent potassium channels (Kv) either by occluding the conduction pore (pore blockers) or by modifying the channel gating properties (gating modifiers). Gating modifiers of Kv channels bind to four equivalent extracellular sites near the S3 and S4 segments, close to the voltage sensor. Phrixotoxins are gating modifiers that bind preferentially to the closed state of the channel and fold into the Inhibitory Cystine Knot structural motif. We have solved the solution structure of Phrixotoxin 1, a gating modifier of Kv4 potassium channels. Analysis of the molecular surface and the electrostatic anisotropy of Phrixotoxin 1 and of other toxins acting on voltage‐dependent potassium channels allowed us to propose a toxin interacting surface that encompasses both the surface from which the dipole moment emerges and a neighboring hydrophobic surface rich in aromatic residues.

Insecticidal peptides from the theraposid spider brachypelma albiceps: an NMR-based model of Ba2

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.

Solution structure of ADO1, a toxin extracted from the saliva of the assassin bug, Agriosphodrus dohrni

ADO1 is a toxin purified from the saliva of the assassin bug, Agriosphodrus dohrni. Because of its similarity in sequence to Ptu1 from another assassin bug, we did not assess its pharmacologic target. Here, we demonstrate by electrophysiologic means that ADO1 targets the P/Q‐type voltage‐sensitive calcium channel. We also determine the solution structure of ADO1 using two‐dimensional NMR techniques, followed by distance geometry and molecular dynamics. The structure of ADO1 belongs to the inhibitory cystine knot (ICK) structural family (i.e., a compact disulfide‐bonded core from which four loops emerge). ADO1 contains a two‐stranded, antiparallel β‐sheet structure. We compare the structure of ADO1 with other voltage‐sensitive calcium‐channel blockers, analyze the topologic juxtaposition of key functional residues, and conclude that the recognition of voltage‐sensitive calcium channels by toxins belonging to the ICK structural family requires residues located on two distinct areas of the molecular surface of the toxins. Proteins 2003.

A Nuclear Magnetic Resonance-Based Structural Rationale for Contrasting Stoichiometry and Ligand Binding Site(s) in Fatty Acid-Binding Proteins

Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.