Multidimensional signatures in antimicrobial peptides

Abstract

Conventional analyses distinguish between antimicrobial peptides by differences in amino acid sequence. Yet structural paradigms common to broader classes of these molecules have not been established. The current analyses examined the potential conservation of structural themes in antimicrobial peptides from evolutionarily diverse organisms. Using proteomics, an antimicrobial peptide signature was discovered to integrate stereospecific sequence patterns and a hallmark three-dimensional motif. This striking multidimensional signature is conserved among disulfide-containing antimicrobial peptides spanning biological kingdoms, and it transcends motifs previously limited to defined peptide subclasses. Experimental data validating this model enabled the identification of previously unrecognized antimicrobial activity in peptides of known identity. The multidimensional signature model provides a unifying structural theme in broad classes of antimicrobial peptides, will facilitate discovery of antimicrobial peptides as yet unknown, and offers insights into the evolution of molecular determinants in these and related host defense effector molecules.

Fig. 1.

Enantiomeric sequence patterns in cysteine-containing antimicrobial peptides. Representatives are, in descending order (peptide name, GenBank GI accession code): α-defensins (HNP-3, 229858); β-defensins (mBD-8, 15826276); insect defensins (phormicin, 118432); insect CS-αβ peptides (drosomycin, 2780893); plant CS-αβ peptides (Ah-AMP-1, 6730111); crustacean CS-αβ peptides (MGD-1, 12084380); protegrins (protegrin-1, 3212589); big defensin (2493577); gaegurins (gaegurin-1, 1169813); tachyplesins (tachyplesin-1, 84665); polyphemusins (polyphemusin-1, 130777); mytilins (mytilin A, 6225740); gomesin (20664097); thanatin (6730068); and AFP-1 (1421258). Sequences are shown in their conventional dextromeric orientations unless indicated as levomeric (levo). The GXC or CXG-C motif is highlighted [glycine (G), orange; cysteine (C), yellow] within primary sequences corresponding to the γ-core motif (outlined). Conserved cysteine residues beyond the γ-core are shaded gray.

Fig. 2.

(A-H) Conservation of 3D structure among disulfide-stabilized antimicrobial peptides. Comparisons (peptide name, PDB ID code, source genus, common name; rmsd) are between Ah-AMP-1 (1BK8, Aesculus, horse chestnut tree) and protegrin-1 (PRO-1,1PG1, Sus, domestic pig; rmsd 1.2 Å; A and B); drosomycin (DRO, 1MYN, Drosophila, fruit fly; rmsd 1.2 Å; C and D); HNP-3 (1DFN, Homo, human; rmsd 3.2 Å; E and F); and magainin-2 (MAG-2, 2MAG, Xenopus, frog; rmsd 2.6 Å; G and H). A, C, E, and G use clustal secondary structure coloration (gold, β-sheet; red, α-helix; blue, turn). B, D, F, and H use the clustal polarity-2 color scheme (hydrophobic, gray; hydrophilic, purple). Oxidized cysteine residues (cystine) are colored gray, indicating hydrophobicity. Disulfide bonds are indicated as dotted yellow lines in A-H. Proteins were visualized by using protein explorer (26). (I-L) Absence of the γ-core signature in nonantimicrobial peptides. Representative comparisons are between the antimicrobial peptide Ah-AMP-1 and nonantimicrobial peptides: allergen-5 (2BBG, Ambrosia, ragweed; rmsd 6.5 Å; I); metallothionein II (1AOO, Saccharomyces, yeast; rmsd 5.3 Å; J); transforming growth factor α (3TGF, Homo, human; rmsd 4.7 Å; K); and ferredoxin (2FDN, Clostridium, bacterium; rmsd 7.4 Å; L). Nonantimicrobial peptides (blue) are shown in maximal alignment to Ah-AMP-1 (gray). Formatting is same as in A-H.

Fig. 3.

Conservation of the γ-core motif among disulfide-containing antimicrobial peptides. The conserved γ-core motif (red) is indicated with corresponding sequences (GXC or CXG-C motifs are denoted in red text). Example molecules, peptide (source), are organized into four structural groups relative to the γ-core. γ Group: protegrin-1 (1PG1), gomesin (1KFP), tachyplesin-1 (1MA2), RTD-1 (1HVZ), thanatin (8TFV), and hepcidin (1M4F). γ-α Group: sapecin (1L4V), insect defensin A (1ICA), heliomycin (1I2U), drosomycin (1MYN), MGD-1 (1FJN), and charybdotoxin (2CRD). β-γ Group: HNP-3 (1DFN), RK-1 (1EWS), BNBD-12 (1BNB), HBD-1 (1E4S), HBD-2 (1E4Q), and mBD-8 (1E4R). β-γ-α Group: Ah-AMP-1 (1BK8), Rs-AFP-1 (1AYJ), Ps-Def-1 (1JKZ), γ-1-H-thionin (1GPT), γ-1-P-thionin (1GPS), and brazzein (1BRZ). Protegrin, gomesin, tachyplesin, RTD-1, and thanatin γ-core sequences (γ Group) are depicted in levomeric orientation. Other formatting is as in Fig. 2.

Fig. 4.

Iterations of the 3D γ-core motif. Amino acid consensus patterns of the three γ-core sequence isoforms are shown. Coloration represents the most common residue (>50% frequency) at a given position, as adapted from the RASMOL (raster-pixel array molecule) schema: cysteine (C), yellow; glycine (G), orange; lysine or arginine, royal blue; serine or threonine, peach; leucine, isoleucine, alanine, or valine, dark green; aromatic, aqua; and variable positions (<50% consensus), gray.

Fig. 5.

Structural validation of the multidimensional signature model. Structures: brazzein [1BRZ, Pentadiplandra,j'oblie berry (11); A, D, and G], charybdotoxin [2CRD, Leiurus, scorpion (27); B, E, and H)], tachyplesin I (1MA2, Tachypleus, horseshoe crab; C, F, and I). Respective γ-core motifs are highlighted in red (G-I; as in Fig. 3). Formatting is as in Fig. 2.

Fig. 6.

Functional validation of the multidimensional signature model. Radial diffusion assays (10) were conducted by using defensin HNP-1 (HNP); brazzein (BRZ); charybdotoxin (CTX); or metallothionein II (MTL). Histograms express mean (±SD; minimum n = 2) zones of complete (blue) or incomplete (yellow) growth inhibition. Note differences in scale.