title: Solution structures of the cis- and trans-Pro30 isomers of a novel 38-residue toxin from the venom of Hadronyche Infensa sp. that contains a cystine-knot motif within its four disulfide bonds.
abstract: The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-Hi:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-Hi:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing the presence of a triple-stranded antiparallel beta sheet consistent with the inhibitor cystine-knot (ICK) motif.
authors.affiliation: Institute for Molecular Bioscience, ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane QLD 4072, Australia.
authors.forename: K Johan
authors.initials: KJ
authors.lastname: Rosengren
authors.forename: David
authors.initials: D
authors.lastname: Wilson
authors.forename: Norelle L
authors.initials: NL
authors.lastname: Daly
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000737:chemistry*; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry*; D007536:Isomerism; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013111:Spider Venoms / Q000737:chemistry* / Q000302:isolation & purification; D013112:Spiders
chemical_list: D017931:DNA Primers; D004220:Disulfides; D009498:Neurotoxins; D012996:Solutions; D013111:Spider Venoms; C451900:atracotoxin-Hi-OB4219; D003553:Cystine