Abstract
Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin–lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin–phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption.
Highlights
Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens
Since binding of phosphatidic acid (PA) has been previously shown to be important for membrane permeabilization and fungal cell killing by plant defensins[19,23,25], we examined the ability of Nicotiana alata defensin 1 (NaD1) to bind PA and form oligomeric complexes using chemical crosslinking or negative stain TEM (Fig. 1a,b)
The ten dimers are arranged in a configuration that results in the formation of a mildly curved, carpet-like structure which we refer to as the membrane disruption complex (MDC), with two such 20-mers forming a large 40-meric ellipsoid complex with a total of 28 PA molecules (Fig. 3b,c)
Summary
The direct dimer–dimer interactions are primarily via cooperative binding of PA (discussed below), and a single hydrogen bond between Glu[6] from one dimer and the backbone of Lys[17] from an adjacent dimer (Supplementary Figure 5a) This interface is designated as type A. The two tetramers within the central arc are not stabilized by PA-mediated interactions, but instead associate via a more extensive hydrogen bond network consisting of Glu6–Lys[17], and Arg[40] to the backbones of Cys[34] and Ser[35] (Supplementary Figure 5b) This interface is designated as type B. We examined C. albicans LTUMC001 and an additional two clinical isolates ATCC10231 and ATCC90028, which all displayed significant PI uptake when treated with low μM concentrations of wild-type NaD1 (Fig. 7b) and substantial reductions of colony forming units (Supplementary Figure 7). We confirmed the PI uptake results by performing live confocal laser scanning microscopy, where we observed rapid PI uptake in C. albicans (LTUMC001) when treated with wild-type NaD1, whereas both NaD1(K36E) and NaD1(R39A) showed substantially delayed and reduced PI uptake (Fig. 7d)
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