1Cooperative Research Centre for Tropical Plant Pathology, University of Queensland, Brisbane, Queensland 4072, Australia; 2Centre for Drug Design and Development, University of Queensland, Brisbane 4072, Australia

Background and objectives
MiAMP1 is a low molecular weight antimicrobial peptide isolated from the nut kernel of Macadamia integrifolia spp. [1]. The peptide is highly basic, with an estimated pI of 10.1 and a mass of 8.1 kDa, and contains 76 amino acids of which six are disulphide bound cysteines. The amino acid sequence and cysteine spacing of MiAMP1 make this peptide the first described member of a new family of antimicrobial peptides. The purified peptide showed inhibitory activity against a variety of microbial phytopathogens. However, the inhibitory activity of MiAMP1 is diminished in the presence of divalent cations (Ca2+). Antimicrobial peptides when expressed in transgenic plants have the potential to provide resistance to plant pathogens, but it has been proposed that the sensitivity of antimicrobial peptides to cations may diminish the applications in transgenic plants. Therefore there is a need to develop peptides with higher potency and with reduced sensitivity to cations. The objective of this study was to use the recently determined 3D structure of the plant antimicrobial peptide MiAMP1 to produce variant peptides with altered surface properties. These studies have shown that it is possible to make substantial improvements in the in vitro activity of this peptide using a bioengineering approach.

Results and conclusions
The global 3D structure of MiAMP1 has recently been determined using NMR spectroscopy and is composed of six strands of beta-sheet linked together by various external loops. When the structure is viewed with regard to charge distribution the majority of the surface basic residues are grouped together on one face, with the opposite face composed primarily of neutral and hydrophobic residues. This separation of charged from uncharged residues in antimicrobial peptides is believed to be important for their mode of action. Variants of MiAMP1 were designed based on a combination of the 3D structure and solvent exposure of specific residues. A PCR-based method was used to mutagenize the coding sequence of MiAMP1 which was then cloned into a modified pET vector and expressed in E. coli. As was expected, removal of basic residues from the surface of the peptide resulted in a decrease or complete loss of activity. Likewise, two surface tyrosine residues were also found to be necessary for full antimicrobial activity. Tyrosines have been previously implicated in the mode of action of thionins believed to play a crucial role in binding to the target cell. Increasing the basic nature of the peptide was performed by either the replacement of neutral with basic residues or via the removal of the one surface-exposed acidic residue. In a few cases these caused slight increases in activity in low ionic strength media, but most importantly several of these variants improved the tolerance of the peptide to divalent cations. The most dramatic changes in biological activity occurred resulting from changes to residue 54, a histidine, present in a region composed primarily of neutral and hydrophobic residues. Residue 54 was changed to either a valine or a lysine: the former to examine the importance of the hydrophobic nature of this face of the peptide, and the latter to see if an increase in the basic charge of this residue affected activity. The valine substitution was six times more potent against Sclerotinia sclerotiorum and Alternaria brassicicola, while the lysine substitution was most effective against Fusarium oxysporum exhibiting a 5 increase in bioactivity, indicating some level of specificity. Both of these substitutions resulted in 5-20 times greater tolerance of divalent cations as compared to that of the native peptide. Experiments are planned to express the more potent variants in transgenic plants to determine if they lead to enhanced resistance to fungal pathogens.

1. Marcus JP, Goulter KC, Green JL et al., 1997. European Journal of Biochemistry 244, 743-49.