An Unusual Helix-Turn-Helix Protease Inhibitory Motif in a Novel Trypsin Inhibitor from Seeds of Veronica (Veronica hederifolia L.)

Abstract

The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent K i and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two ␣ helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.Plant seeds are rich sources of proteinaceous proteinase inhibitors. These are believed to form a wide spectrum defense mechanism against fungal pathogens and invertebrate pests but may also play a role in the regulation of metabolism and act as storage proteins (1). A diverse range of medicinal properties have also been associated with many of these inhibitors, including anti-human immunodeficiency virus activity (2), hemolytic activity (3, 4), and inhibition of neurotensin binding (5) On the basis of their amino acid sequence and target proteinases, plant proteinase inhibitors have been classified into a number of families (6). The two best characterized are the Kunitz and Bowman-Birk families. The Kunitz soybean trypsin inhibitor was the first to be extensively characterized (7) and is an all ␤-sheet protein of 20 kDa. One exposed surface loop and the N terminus of the protein interact closely with the trypsin molecule, whereas the vast majority of the inhibitor forms no direct contacts with its inhibitory target. The Bowman-Birk family of serine protease inhibitors (reviewed in Ref. 8) are smaller proteins of ϳ8 kDa that contain seven conserved disulfide bridges. They have two reactive sites that are able to bind to the active sites of a number of serine proteases including trypsin and chymotrypsin from human, animal, and insect sources. The reactive site residues of the inhibitor lie within a ␤-hairpin region (stabilized by a disulfide bond), which enables them to be presented in the same conformation as the normal peptide substrate (9). Like the Kunitz family of serine protea...

Genes encoding 4‐Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: multimodular structural organization, instraspecific variability, distribution and role in defence

“…The cysteine motif of these peptides may therefore be presented as: C1XXXC2-X(n)-C3XXXC4. The same cysteine pattern is shared by AMPs from two other cereals, maize (MBP-1) [15] and barnyard grass Echinochloa crus-galli (EcAMP1) [16], a dicotyledonous plant the Queensland nut Macadamia integrifolia (MiAMP2) [17], trypsin inhibitors from the ivy-leaved speedwell Veronica hederifolia (VhTI) [18] and buckwheat Fagopyrum esculentum (BWI-2b and BWI-2c) [19,20]. Moreover, we established the disulfide connectivity in Tk-AMP-X2, which was found to be identical to that in EcAMP1, VhTI, BWI-2b and BWI-2c.…”

“…The peptide was cleaved at Met residues by cyanogen bromide, and the resulting mixture was analyzed by MS. Two fragments were identified with molecular masses of 1628 and 1831 Da. The latter corresponds to the middle part of the peptide (residues [9][10][11][12][13][14][15][16][17][18][19][20][21][22] containing the 'inner' Cys 9 -Cys 21 disulfide bond. The former corresponds to the two flanking parts (residues 1-8 and 23-28) held together by the 'outer' Cys 5 -Cys 25 disulfide bond.…”

Novel Detection Methods Used in Conjunction with Affinity Chromatography for the Identification and Purification of Hydrolytic Enzymes or Enzyme Inhibitors from Insects and Plants

“…At that time no homology with known inhibitors was found. Both the native inhibitor and corresponding especially synthesized peptide have been crystallized and their three-dimensional structures were determined (Conners et al, 2007). It was found that this inhibitor contains an unusual for inhibitors helix-turn-helix proteinase inhibitory motif.…”

“…Affinity matrices using proteinases (trypsin, chymotrypsin and subtilisin) or proteinase inhibitors immobilized on CNBr-activated Sepharose or acrylamide or agarose gels were used for purification of proteinase inhibitors from plants and proteinases from insects (Conners et al, 2007;Konarev 1986a, Konarev et al, 2000, 2002a, 2008, 2011. For analytical work, certain types of inhibitor or proteinase were pre-absorbed from protein mixtures by modification of the method of Hejgaard et al (1981) to facilitate the identification of other types of inhibitors or proteinases.…”

“…Its use in conjunction with detection methods that can quickly and easily identify components of proteins mixtures with the sought-after activity can appreciably simplify the search and purification of novel enzymes or enzyme inhibitors, especially those with low or non-typical substrate specificities. Advances have been made in fractionation and detection methods by many laboratories but we describe below approaches used by us including the use of affinity chromatography in combination with novel detection methods to identify and purify novel forms of insect and fungal proteinases and also proteinase inhibitors from plants (Conners et al, 2007;Konarev, 1985Konarev, , 1986Konarev, , 1990aKonarev & Fomicheva, 1991a;Konarev et al, 1999aKonarev et al, , 1999bKonarev et al, , 2000Konarev et al, , 2002aKonarev et al, , 2004Konarev et al, , 20082011;Luckett et al, 1999). In some cases these have been used for the analysis of the diversity of amylase and proteinase inhibitors in various plant taxa (Konarev 1982a(Konarev , 1986b(Konarev , 1987b …”