Characterization of Hydroxyproline-Containing Hairpin-Like Antimicrobial Peptide EcAMP1-Hyp from Barnyard Grass (Echinochloa crusgalli L.) Seeds: Structural Identification and Comparative Analysis of Antifungal Activity

Abstract

Herein, we describe a modified form of the antimicrobial hairpin-like peptide EcAMP1, isolated from barnyard grass (E. crusgalli) seeds, which is structurally characterized by a combination of high-pressure liquid chromatography, mass spectrometry, and automated Edman sequencing. This derivate has a single amino acid substitution (Pro19Hyp) in the second α-helical region of the molecule, which is critical for the formation of the hydrophobic core and the secondary structure elements. Comparing the antifungal activity of these two peptides, we found that the modified EcAMP1-Hyp had a significantly weaker activity towards the most-sensitive plant pathogenic fungus Fusarium solani. Molecular dynamics simulations and in vitro binding to the commercial polysaccharides allowed us to conclude that the Pro-19 residue is important for binding to carbohydrates located in the spore cell wall and it chiefly exhibits a fungistatic action representing the hyphal growth inhibition. These data are novel and significant for understanding a role of α-hairpinins in plant immunity.

Rational Design of Plant Hairpin-like Peptide EcAMP1: Structural–Functional Correlations to Reveal Antibacterial and Antifungal Activity

“…This confirms a critical role of tryptophan residue located in the β-hairpin in antifungal and antibacterial activity. Moreover, the unusual natural form of the EcAMP1 peptide consisting of a substitution from proline to hydroxyproline at the 19th position demonstrated a weaker activity against a model plant pathogenic fungus F. solani [ 19 ]. In the case of EcAMP1-X4, the removal of six C-terminal amino acid residues concerns the second α-helix and might lead to a destabilization of the molecule structure.…”

“…Chemical synthesis of the truncated EcAMP1-X1 and EcAMP1-X2 was carried out as described earlier [ 19 ] with modifications [ 33 ]. Briefly, solid-phase peptide synthesis was performed on an automatic peptide synthesizer (Agilent Technologies, Santa Clara, CA, USA) based on the Gilson automated liquid handler system (Gilson Scientific Ltd., Dunstable, UK), according to Gilson application note 228 (Gilson Scientific Ltd., Dunstable, UK).…”

“…First of all, EcAMP1 an antifungal α-hairpinin from barnyard grass ( E. cruss-galli ) seeds, is known to internalize inside fungal conidia without plasma membrane disruption [ 18 ]. Amino acid substitution Pro19Hyp leads to a decrease in antifungal activity and carbohydrate binding [ 19 ]. In addition, the loss of C-terminal random coil in EcAMP2 leads to a complete loss of peptide activity [ 15 ].…”

Defense Peptides From the α-Hairpinin Family Are Components of Plant Innate Immunity

“…Considering this data and low antifungal activity, it was concluded that the tails of the molecule contributed either through direct interaction with the fungi or through stabilization of the helical structure (Slavokhotova et al, 2014b). Rogozhin et al (2018b) identified several structural elements of α-hairpinins that play a key role in their biological activity. According to their experiments and the literature data, the first crucial element is a β-hairpin structure (10-13 amino acid residues) that connects two α-helices.…”

“…The second element is a mini-cluster of positively charged amino acid residues necessary for interaction with negatively charged carbohydrate components and polymers of fungal cell walls. And the third element is the presence of at least one hydrophobic residue (e.g., tryptophan) pivotal for binding with components of fungal cell membranes, including sphingolipids or ergosterols (Rogozhin et al, 2018b).…”