Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

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

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…Similar PI(4,5)P 2 -binding mechanism was also described for the tomato defensin TPP3, which binds specifically to PI(4,5)P 2 and shares the conserved cationic grip binding pocket as NaD1 to mediate cell lysis. 70 It should be noted that the cationic loop region identified as the PA binding site in MtDef4, is conserved among many plant defensins 119,120 and interestingly, the equivalent region (between β2 and β3 strands) was also found to be the PI (4,5)P 2 binding site for both NaD1 and TPP3, 69,70 indicating that this region is functionally conserved to bind phospholipid. Intriguingly, the ability of these plant innate defense molecules to induce membrane permeabilization via binding to inner membrane PI(4,5)P 2 and forming oligomers resembles the aforementioned PI(4,5)P 2 -dependent targeting and oligomerization by MLKL in mammalian cells undergoing necroptosis.…”

“…49 Recently, it was demonstrated that the plant defensin NaD1, a CAP isolated from the ornamental tobacco Nicotiana alata, can selectively induce membrane lysis (primary necrosis) of mammalian tumor cells through binding PI(4,5)P 2 at the inner leaflet of the plasma membrane at low micromolar concentrations. 69 Although how NaD1 enters the tumor cell and gains access to the intracellular phospholipid code is unclear, the ability of NaD1 to form a multimeric complex with the phosphatidylinositol head group of PI(4,5)P 2 suggests the possibility of NaD1 acting as a phospholipid detector that can mask the cellular function of PI(4,5)P 2 , discussed further below. 69 More recently, the closely related tomato defensin, TPP3, was also shown to induce PI(4,5)P 2 -mediated tumor cell permeabilization via a similar mechanism.…”

“…69 Although how NaD1 enters the tumor cell and gains access to the intracellular phospholipid code is unclear, the ability of NaD1 to form a multimeric complex with the phosphatidylinositol head group of PI(4,5)P 2 suggests the possibility of NaD1 acting as a phospholipid detector that can mask the cellular function of PI(4,5)P 2 , discussed further below. 69 More recently, the closely related tomato defensin, TPP3, was also shown to induce PI(4,5)P 2 -mediated tumor cell permeabilization via a similar mechanism. 70 Exploiting the phospholipid code during host-pathogen interaction.…”

“…48 Likewise, the aforementioned ornamental tobacco defensin, NaD1, which binds PI(4,5)P 2 as well as a range of other phosphorylated phosphatidylinositol species, also exhibits antifungal activity against filamentous fungi, in part mediated by phospholipid binding. 69 NaD1 was solved by X-ray crystallography in complex with PI(4,5)P 2 , revealing the formation of a dimeric 'cationic grip' PI(4,5)P 2 binding pocket, repeated to form an extended oligomeric arch. This interaction was proposed to induce membrane destabilization of target cells, with sitedirected mutagenesis of key lipid-binding residues confirming the requirement of PI(4,5)P 2 for efficient membrane lysis of both Fusarium oxysporum as well as mammalian tumor cells 69,118 (Figure 2i).…”

“…69 NaD1 was solved by X-ray crystallography in complex with PI(4,5)P 2 , revealing the formation of a dimeric 'cationic grip' PI(4,5)P 2 binding pocket, repeated to form an extended oligomeric arch. This interaction was proposed to induce membrane destabilization of target cells, with sitedirected mutagenesis of key lipid-binding residues confirming the requirement of PI(4,5)P 2 for efficient membrane lysis of both Fusarium oxysporum as well as mammalian tumor cells 69,118 (Figure 2i). Similar PI(4,5)P 2 -binding mechanism was also described for the tomato defensin TPP3, which binds specifically to PI(4,5)P 2 and shares the conserved cationic grip binding pocket as NaD1 to mediate cell lysis.…”