Several NMR structures of anti-fungal peptide were reported in recent years. Their coordinates were deposited to the Brookhaven Protein Data Bank (PDB). The anti-fungal peptide (PDB code 1AFP) from Aspergillus giganteus consists of 51 amino acid residues among them are 12 Lysines. The activity of this peptide is the inhibition of the growth of a variety of filamentous fungi. It has no effect on the growth of mammalian cells, yeast and eubacteria. The structural feature of this peptide is a compact beta-barrel formed by five anti-parallel beta-strands and stabilized by four internal disulfide bridges [Campos-Olivas et al., 1995]. Interestingly, another anti-fungal peptide isolated from radish (Raphanus sativus L.) seeds (1AYJ) poses the same sequence length and same number of disulfide bridges as 1AFP. However, no sequence similarity exists between these two peptides and the folding unit of this peptide is not a beta-barrel, but a cysteine stabilized alpha-beta motif [Fant et al., 1998]. This motif of three anti-parallel beta-strands and an alpha-helix connected by three disulfide bridges has been found in the scorpion toxin family. The biological activity of 1AYJ reduces elongation of fungal hyphae and increases hyphal branching. A third peptide Drosomycin induced by the fruit fly Drosophila melanogaster exhibits a potent activity against filamentous fungi, but inactive against bacteria. This peptide is 7 residue shorter than the above two. Yet, the number of disulfide bridges remains the same. The NMR structure of this peptide (1MYN) reveals that it also belongs to the cysteine stabilized alpha-beta motif [Landon et al., 1997]. The anti-fungal peptide we reported seems not belong to the above two folding categories due to the small size in sequence length and the number of the disulfide bridges.
A small cysteine-rich protein with antimicrobial activity was isolated from pokeweed (Phytolacca americana) seeds and purified to homogeneity. The protein inhibits the growth of several filamentous fungi and gram-positive bacteria. The protein was highly basic, with a pI higher than 10. The entire amino acid sequence of the protein was determined to be homologous to antimicrobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. americana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and sequenced. The deduced amino acid sequence shows the presence of a signal peptide at the amino terminus, suggesting that the protein is synthesized as a preprotein and secreted outside the cells. The chromosomal gene shows the presence of an intron located within the region encoding the signal peptide. Southern hybridization showed that there was small gene family encoding Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, and was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted into the environment of the seeds during germination, and may create an inhibitory zone against soil-borne microorganisms. The disulfide bridges of Pa-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicates that the protein has a small cystine-knot folding, a positive patch, and a hydrophobic patch.