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 α-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.

FIGURE 1.

Purification of EcAMP1. A, size-exclusion chromatography of the 500 mm NaCl fraction from heparin-Sepharose on a Sephacryl S-100 HR column. The fraction containing EcAMP1 is shown with a bar. B, RP-HPLC on a Luna C₁₈ column. The peak corresponding to EcAMP1 is indicated.

FIGURE 2.

EcAMP1 amino acid sequence alignment with similar peptides. Sequences of the following peptides are shown: EcAMP1 from E. crus-galli (P86698; this work); antimicrobial peptides MBP-1 from Z. mays (P28794 (31)) and 2d from M. integrifolia (Q9SPL5 (32)); C2 peptide processed from the PV100 protein in Cucurbita maxima (Q9ZWI3 (54)); ribosome-inactivating peptide luffin P1 from Luffa aegyptiaca (P56568 (55)); trypsin inhibitors VhTI from V. hederifolia (P85981 (34)) and BWI-2b from Fagopyrum esculentum (no UniProt entry (33)). Each peptide length is indicated in the right column. Cysteine residues are in bold and shaded. Disulfide pairing has been determined for EcAMP1, VhTI, and BWI-2b.

FIGURE 3.

Distribution of EcAMP1 in the spores of F. solani. Interactions of EcAMP1 with F. solani spores were visualized by confocal laser scanning microscopy. Confocal fluorescence (left panels) and transmitted light (right panels) images are presented. Incubation time and peptide concentration (2 μm EcAMP1-TMR or 4 μm equimolar mixture of EcAMP1 and EcAMP1-TMR) are indicated. Experimental conditions of the confocal image measurements were fixed, and images can be directly compared (within columns) in intensity of peptide binding to spores and intracellular accumulation.

FIGURE 4.

Amino acid sequence of EcAMP1, sequential connectivities, and additional data collected for secondary structure identification. The sequence of EcAMP1 is displayed at the top and bottom. The sequential NOE connectivities d_NN, d_αN, and d_βN are indicated with black bars of a thickness proportional to the volume of the NOE cross-peaks. The medium range connectivities d_αN(i,i + 2), d_αN(i,i + 3), d_αβ(i,i + 3), and d_αN(i,i+4) are shown by lines starting and ending at the positions of the residues related by NOEs. The NOE information on some regions was restricted by overlapping cross-peaks. In the row H-D_ex, slowly hydrogen-deuterium exchanging amide groups at pH 3.4 and 20 °C with estimated half-exchange times 0.5 h < t½ < 1 h (t½ > 1 h) are depicted by open (closed) circles. In the row CSI, bar height is proportional to chemical shift index given as the difference between the actual ¹Hα chemical shift of an EcAMP1 residue and a typical random coil chemical shift of this residue. Pronounced negative value of the chemical shift index indicates helical conformation of a residue. In the row H-bonds, closed and open boxes (triangles) depict donors of i,i+4 (i,i+3) backbone hydrogen bonds identified by MOLMOL for more than 17 and 9 structures of the NMR ensemble, respectively. Locations of secondary structure elements calculated with the STRIDE program (56) are given.

FIGURE 5.

Spatial structure of EcAMP1. A, ribbon diagram of the 20 best CYANA structures superimposed on the backbone atoms of residues 7–30. Buried side chains are displayed and labeled; those forming the hydrophobic core (Cys-7, aliphatic part of Arg-8, Cys-11, Met-12, Pro-19, Val-22, Cys-25, Val-26, and Cys-29) are colored green, and the side chain of His-15 is colored magenta. The side chain of Trp-20 is labeled and colored blue. Disulfide bridges are displayed as yellow sticks. The backbone H^N atom of Cys-7 involved in a transient hydrogen bond with a C-terminal carboxylate is depicted by a red sphere. The N and C termini are labeled. B, as compared with A, the structure is rotated as depicted in between the panels, and the side chains of Glu-18 and Arg-21 forming a salt bridge are shown, labeled, and colored red.