title: Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides.
abstract: A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.
authors.affiliation: M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997 Moscow, Russia.
authors.forename: Peter B
authors.initials: PB
authors.lastname: Oparin
authors.forename: Konstantin S
authors.initials: KS
authors.lastname: Mineev
authors.forename: Yakov E
authors.initials: YE
authors.lastname: Dunaevsky
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Arseniev
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Belozersky
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Grishin
authors.forename: Tsezi A
authors.initials: TA
authors.lastname: Egorov
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Vassilevski
mesh_terms: D000595:Amino Acid Sequence; D056148:Chromatography, Reverse-Phase; D019612:Fagopyrum / Q000737:chemistry* / Q000502:physiology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors