title: Atypical reactive center Kunitz-type inhibitor from the sea anemone Heteractis crispa.
abstract: The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the K(i) of InhVJ to trypsin and α-chymotrypsin (7.38 × 10(-8) M and 9.93 × 10(-7) M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Irina
authors.initials: I
authors.lastname: Gladkikh
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Margarita
authors.identifier: 0000-0003-3157-0930
authors.initials: M
authors.lastname: Monastyrnaya
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Elena
authors.initials: E
authors.lastname: Leychenko
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Elena
authors.initials: E
authors.lastname: Zelepuga
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Victoria
authors.initials: V
authors.lastname: Chausova
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Marina
authors.identifier: 0000-0002-2395-0485
authors.initials: M
authors.lastname: Isaeva
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Anastyuk
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10, Miklukho-Maklaya Str., Moscow 117997, Russian Federation.
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Andreev
authors.affiliation: Laboratory of Toxicology, University of Leuven (K.U. Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven B-3000, Belgium.
authors.forename: Steve
authors.identifier: 0000-0003-0504-5702
authors.initials: S
authors.lastname: Peigneur
authors.affiliation: Laboratory of Toxicology, University of Leuven (K.U. Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, Leuven B-3000, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Tytgat
authors.affiliation: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, 159, Pr. 100 let Vladivostoku, Vladivostok 690022, Russian Federation.
authors.forename: Emma
authors.initials: E
authors.lastname: Kozlovkaya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D012615:Sea Anemones / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D014361:Trypsin Inhibitors