Novel STAT1 Alleles in Otherwise Healthy Patients with Mycobacterial Disease

The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding

A novel potassium channel blocking toxin from the scorpion Pandinus imperator: A 1H NMR analysis using a nano-NMR probe.

International audienceThe three-dimensional solution structure of a novel peptide, Pi 1, purified from the venom of the scorpion Pandinus imperator and specific for potassium channels was determined by homonuclear proton NMR methods at 500 MHz from nanomole amounts of compound. P. imperator toxin is a voltage-dependent potassium channel specific peptide capable of blocking the shaker B K+ channels expressed in Sf9 cells in culture (Spodoptera frugiperda cell line no. 9) and displacing labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has only 35 amino acid residues but is stabilized by four disulfide bridges (Cys4-Cys25, Cys10-Cys30, Cys14-Cys32, and Cys20-Cys35) instead of three commonly found in small potassium channel toxins. A detailed nuclear magnetic resonance structure of this protein was obtained using a nano-NMR probe and a combination of two-dimensional proton NMR experiments. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the solution conformation of the toxin. The structure is organized around a short alpha-helix spanning residues Ser8-Thr18 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys10-Cys30 and Cys14-Cys32. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys32 with a tight turn at Arg28-Met29 in contact with the N-terminal fragment Leu1-Cys4. Comparison between the 3D structure of Pi 1 and those of other structurally and functionally related scorpion toxins is presented

1H NMR structural analysis of novel potassium blocking toxins using a nano-NMR probe

International audienc

Pi7, an orphan peptide from the scorpion Pandinus imperator: a 1H-NMR analysis using a nano-NMR Probe.

International audienceThe three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specific receptor has been found yet, was determined by two-dimensional homonuclear proton NMR methods from a nanomole amount of compound using a nano-nmr probe. Pandinus imperator peptide 7 does not block voltage-dependent K(+)-channels and does not displace labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has 38 amino acid residues and, similarly to Pi1, is stabilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, and Cys22-Cys37). In addition, the lysine at position 26 crucial for potassium-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equivalent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are occupied by two acidic residues Asp and Glu, respectively. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the peptide. The disulfide-bridge topology was established using distance restraints allowing ambiguous partners between S atoms combined with NMR-derived structural information. The structure is organized around a short alpha-helix spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys12-Cys32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact with the N-terminal fragment Ile4 to Cys6

Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase.

International audienceThe structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr

Solution structure of Cn5, a crustacean toxin found in the venom of the scorpions Centruroides noxius and Centruroides suffusus suffusus.

International audienceThe crustacean toxin Cn5 from Centruroides noxius Hoffmann and peptide Css39.8 from Centruroides suffusus suffusus scorpion venoms are identical peptides, as confirmed by amino acid sequence of purified toxins and by DNA sequencing of the two respective cloned genes. Therefore in this communication they will be simply named Cn5. Cn5 is a 66 amino acid long peptide with four disulfide bridges, formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure). This peptide is non-toxic to mammals but deadly to arthropods (LD(50) 28.5 mg/g body weight of crayfish). Its three-dimensional structure was determined by NMR using a total of 965 meaningful distance constraints derived from the volume integration of the 2D NOESY spectra. The Cn5 structure displays a mixed alpha/beta fold stabilized by four disulfide bridges, with a kink induced by a cis-proline in its C-terminal part. Cn5 electrostatic surface is compared to that of Cn2 toxin toxic to mammals. The local differences produced by additional or substituted residues that would influence toxin selectivity towards mammalian or crustacean Na(+) channels are discussed