Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant

Mark R. Bleackley, Jennifer A. E. Payne, Brigitte M. E. Hayes, Thomas Durek, David J. Craik, Thomas M. A. Shafee, Ivan K. H. Poon, Mark D. Hulett, Nicole L. van der Weerden, Marilyn A. Anderson

The plant defensin NaD1 is a potent antifungal molecule that also targets tumour cells with high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity on fungiand no activity on tumour cells. All plant defensins have a common tertiary structure known as a cysteine stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulphide bonds. The chimeras were produced by replacing loops 1-7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5 bis-phosphosphate (PI(4,5)P2) and is essential for the potent cytotoxic effect of NaD1 on tumour cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumour cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules even though they bound poorly to PI(4,5)P2 suggesting additional mechanisms operate against fungal cells. Unexpectedly the L1B loop swap chimera was ten times more active than NaD1 on filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to natural variants.