Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

Ivan Kh Poon,Julie A White,Prem K Veneer,Gemma F Ryan,Marc Kvansakul,Fung T Lay,Nicole L Van Der Weerden,Jennifer Ae Payne,Christopher G Adda,Amy A Baxter,Mark D Hulett,Grant D Mills,Thanh Kha Phan,Marilyn A Anderson

doi:10.7554/elife.01808

Abstract

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001.

Highlights

Host defense peptides, which include cationic antimicrobial peptides (CAPs), are a group of innate immune molecules produced by essentially all plant and animal species that act as a first line of defense against microbial invasion
We have identified the cellular phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) as a key ligand that is recognized during membrane permeabilization of fungal and mammalian plasma membranes
Membrane lipids represent an attractive target for NaD1; we investigated whether NaD1 interacts with cellular lipids using protein–lipid overlay assays based on lipid strips immobilized with 100 pmoles of various biologically active lipids (Poon et al, 2010; Patel et al, 2013)

Summary

Introduction

Host defense peptides, which include cationic antimicrobial peptides (CAPs), are a group of innate immune molecules produced by essentially all plant and animal species that act as a first line of defense against microbial invasion. The defensins are a family of CAPs that are ubiquitously expressed in plants, animals, insects, and fungi that play an important role in innate immune defense against microbial threats (Brogden, 2005; Lay and Anderson, 2005; Hancock and Sahl, 2006; Lai and Gallo, 2009). The sequence variability leads to several biological functions including antimicrobial activity, regulation of plant development, and pollen tube guidance (Carvalho and Gomes, 2009; De Coninck et al, 2013). Even those plant defensins that have been ascribed antifungal activity have large differences in sequence and are likely to act by different mechanisms (van der Weerden and Anderson, 2013). The plant defensins are small (∼5 kDa, 45–54 amino acids), basic, cysteine-rich proteins that display a family-defining disulfide bond array (in a CI–CVIII, CII–CV, CIII–CVI, and CIV–CVII configuration) known as the

Methods

Results

Conclusion