Journal Article

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

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

2014

Overview

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. It is often said that attack is the best form of defense; and the immune systems of plants and animals will often target the cell membranes of microbes and other pathogens in order to defend themselves. Disrupting the cell membrane causes essential contents to leak from the cell, and eventually, the cell will burst and die. Most plants and animals produce small proteins called defensins that kill microbes by attacking their cell membranes. These defensins are thought to either destabilize the cell membrane by coating its outer surface or to insert themselves into the membrane to form open pores that allow vital biomolecules to leak out of the cell. However, the exact mechanism by which defensins attack microbial membranes is not understood. In this study, Poon, Baxter, Lay et al. show that a defensin called NaD1—which was isolated from the ornamental tobacco Nicotiana alata—binds to a molecule from the cell membrane called phosphatidylinositol 4,5-bisphosphate, or PIP2 for short. By working out the three-dimensional structure of this complex, Poon, Baxter, Lay et al. show that it contains 14 PIP2 molecules and 14 NaD1 molecules in an arch-shaped structure and suggest that sequestering large numbers of PIP2 molecules in this way destabilizes the cell membrane of the microbe. These findings raise a number of questions: are there other small proteins that can destabilize cell membranes in a similar manner to defensins? Do the immune systems of other organisms also recognize molecules from microbial cell membranes to trigger this kind of counterattack? Furthermore, since defensins can also kill tumor cells, a better understanding of how they work might also lead to new treatments for cancer and other diseases in humans.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Amino acids / Antimicrobial agents / antimicrobial peptide / Antimicrobial peptides / Biophysics and Structural Biology / Candida albicans / Cationic antimicrobial peptides / Cell Death / Chemical bonds / Crystal structure / Crystallography, X-Ray / Defensins / Defensins - chemistry / Defensins - genetics / Defensins - isolation & purification / Defensins - metabolism / Epithelial Cells - drug effects / Epithelial Cells - physiology / Fungi / fungus / Fusarium - drug effects / Fusarium - physiology / HeLa Cells / Humans / Ligands / Lipids / Lysis / Membranes / Microbial Viability - drug effects / Microscopy / Mutagenesis, Site-Directed / Nicotiana - chemistry / Oligomerization / Peptides / Phosphatidylinositol 4,5-diphosphate / Phosphatidylinositol 4,5-Diphosphate - chemistry / Phosphatidylinositol 4,5-Diphosphate - metabolism / phospholipid / Phospholipids / Plant cells / Protein Binding / Protein Multimerization / protein structure / Site-directed mutagenesis / Software / Tumor cells / X-ray crystallography