The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis

Abstract

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a "cationic grip" conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species. P lant defensins are small (ϳ5 kDa), cysteine-rich, cationic peptides that belong to the broad class of innate defense molecules known as cationic antimicrobial peptides (CAPs). Plant defensins play a major role in plant innate immunity and have been identified in all analyzed plant species to date, as either constitutively expressed or induced defense molecules that are produced in response to pathogenic attack or environmental stress (1). The tertiary structure of all plant defensins is highly conserved, comprising a triple-stranded antiparallel ␤-sheet and a single ␣-helix stabilized by four disulfide bridges, known as the "cysteine-stabilized alpha-beta" or "CS␣␤" motif (2). Despite this conserved three-dimensional structure, there is a high degree of variation in the primary sequence of plant defensins, particularly at intervening loop regions, which are typically important for activity (3).Many plant defensins have antifungal activity, but other functions have been reported, including antibacterial activity, ion channel blocking, protein synthesis inhibition, and trypsin and ␣-amylase inhibition as well as roles in heavy metal tolerance, plant development, and pollen tube guidance (3-5). They can be divided into two classes based on whether or not a C-terminal propeptide (CTPP) (of ϳ33 amino acids) is present (2, 6). This domain is involved in vacuolar targeting and protects the plant cells from phytotoxicity during transit through the secretory pathway (7). Defensins expressed with the additional CTPP domain are kn...

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.…”

“…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. Pathogens commonly hijack and take advantage of host molecules to facilitate their invasion and replication.…”

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

“…The activity against tumor cell lines is mediated by a specific and high-affinity interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. This lipid binding specificity has been described only for the class II defensins from the Solanaceae among plant defensins and is largely mediated through the sequence in loop 5 (10,11,15). Class II defensins have a C-terminal propeptide that directs them to the vacuole, whereas class I defensins lack this sequence and are secreted by default from the plant cell (9).…”

“…The sequence diversity explains the wide range of functions that have been reported for plant defensins, including antibacterial and antifungal activities as well as roles in plant development, sexual reproduction, and metal tolerance (reviewed in reference 5). Some antifungal defensins display activity against tumor cells but not healthy mammalian cells (10,11). However, it remains to be established if the mechanism of action against fungal and mammalian tumor cells is the same.…”

“…The defensin-PI(4,5)P 2 dimer then forms oligomers via a different interface (10). The interaction of NaD1 and TPP3 with PI(4,5)P 2 is proposed to lead to the characteristic blebbing and membrane permeabilization that occurs when tumor cells are treated with these defensins (10,11). Indeed, masking of PI(4,5)P 2 by intracellular expression of a pleckstrin homology domain or treatment with neomycin delayed tumor cell killing by NaD1, supporting the role for PI(4,5)P 2 binding in the activity against tumor cells (10,11).…”