Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli)

Abstract

This study presents purification, activity characterization, and 1 H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized ␣-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.Antimicrobial peptides (AMPs) 4 are a structurally diverse group of generally small, positively charged peptides produced by various living organisms and demonstrating a wide spectrum of antimicrobial activity (1, 2). Natural sources of AMPs range from prokaryotes to higher animals, and their targets include bacteria, fungi, protozoa, and viruses. The mechanism of action of most known AMPs involves their direct or receptor-mediated interaction with microbial membranes (3-5). It has been generally accepted that membrane-disruptive AMPs kill microorganisms by provoking in different ways an increase in plasma membrane permeability. Non-membrane-disruptive peptides have been shown to target cell wall formation or traverse membranes and affect various internal cellular processes, for example, RNA, DNA, and/or protein biosynthesis. Some AMPs can combine disruptive and non-disruptive mechanisms of action (6). Moreover, mechanisms of action of the same peptide may differ depending on the target. Recent studies have also indicated that AMPs are multifunctional molecules; they can interact with host membrane receptors and influence diverse intracellular processes modulating the immune response of the host organism (7,8).Essential variety in detailed mechanisms of action and multifunctionality imply structural diversity among AMPs. The following structural groups are usually recognized: (i) linear peptides that form ␣-helices in contact with membranes; (ii) disulfide-containing with predominance of ␤-structural elements; and (iii) linear non-␣-helix-forming, usually with a high content of certain amino acid residues (1, 2, 9). Most of the approximately 200 AMP spatial structures known at present (see the Antimicrobial Peptide Database v2.26 (10)) fall into one of the first two groups. Further classification is based on unique features in the sequences and/or structures of AMPs. For example, thionins, defensins, nonspecific lipid transfer proteins, and hevein-and knottin-like peptides have been identified in plants (11-13).To characterize the array of AMPs produced by a plant under certain physiological conditions, we have carried out a systematic analysis of these peptides from...

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

“…It was found that this inhibitor contains an unusual for inhibitors helix-turn-helix proteinase inhibitory motif. The subsequent analysis allowed us to reveal that VhTI is a member of wide group of plant peptides with antimicrobial and inhibitory activities found by other authors (Nolde et al, 2011;Park et al, 1997), first characterized by spatial structure. Their amino acid sequences can differ greatly from VhTI but also contain four cysteine residues in configuration nX-CXXXC-nX-CXXXC-nX.…”

Post-Translational Modifications of Natural Antimicrobial Peptides and Strategies for Peptide Engineering

“…In the case of distinctin, one intermolecular disulfide bond occurs between the two peptide chains, leading to an increase in peptide stability (due to the formation of a helix bundle structure in water) but not antimicrobial activity [43]. A recent study provides a novel example for helical AMPs with two disulfide bonds, which lock the two adjacent helices into a hairpin structure [44]. In addition, three disulfide bonds exist in the 10 saposin-like AMPs that form helix bundle structures [10].…”