Binding of phosphatidic acid by NsD7 mediates the formation of helical defensin–lipid oligomeric assemblies and membrane permeabilization

Abstract

Defensins are cationic antimicrobial peptides that serve as important components of host innate immune defenses, often by targeting cell membranes of pathogens. Oligomerization of defensins has been linked to their antimicrobial activity; however, the molecular basis underpinning this process remains largely unclear. Here we show that the plant defensin NsD7 targets the phospholipid phosphatidic acid (PA) to form oligomeric complexes that permeabilize PA-containing membranes. The crystal structure of the NsD7–PA complex reveals a striking double helix of two right-handed coiled oligomeric defensin fibrils, the assembly of which is dependent upon the interaction with PA at the interface between NsD7 dimers. Using site-directed mutagenesis, we demonstrate that key residues in this PA-binding site are required for PA-mediated NsD7 oligomerization and coil formation, as well as permeabilization of PA-containing liposomes. These data suggest that multiple lipids can be targeted to induce oligomerization of defensins during membrane permeabilization and demonstrate the existence of a “phospholipid code” that identifies target membranes for defensin-mediated attack as part of a first line of defense across multiple species.

Structure of the defensin NsD7 in complex with PIP2 reveals that defensin : lipid oligomer topologies are dependent on lipid type

“…S1). We recently showed that NsD7 also forms oligomeric fibrils when binding PA , however, the resultant oligomers adopt a radically different topology compared to the previously determined NaD1–PIP 2 complex . The NsD7–PA oligomer formed a double helical topology that extended throughout the crystal and featured two distinct lipid binding sites per NsD7 dimer compared to only a single site for the previously determined NaD1–PIP 2 complex.…”

“…The NsD7–PA oligomer formed a double helical topology that extended throughout the crystal and featured two distinct lipid binding sites per NsD7 dimer compared to only a single site for the previously determined NaD1–PIP 2 complex. Structure‐guided mutagenesis revealed that the novel phospholipid site not previously observed in NaD1 was critical for NsD7–PA oligomer formation as well as for fungal cell killing , demonstrating that despite their small size, defensins harbor considerable flexibility in their modes of engagement with phospholipids.…”

Phosphatidic acid in membrane rearrangements

“…According to the current view, such motif does not exist; PA effectors use cationic and/or surface exposed hydrophobic residues for binding to PA . Indeed, in the structures of PA‐binding proteins and peptides obtained using X‐ray crystallography and NMR , the binding of positively charged and hydrophobic residues to PA was often observed.…”

“…In the studies of PA–protein interactions, various assays were used. In some of these assays, lipids were immobilized on nitrocellulose filter , coupled to the beads , or organized into liposomes . These assays are well discussed in Refs .…”

Cryo-EM of bacterial pili and archaeal flagellar filaments

“…2). Although highly unusual and unexpected, such a stoichiometric lipoprotein complex was also seen in a defensin-lipid oligomeric assembly [32]. While previously it had been assumed that the subunit-subunit interfaces forming this polymer consisted of hydrophobic α-helices from one subunit packed against hydrophobic α-helices from another subunit, it is now clear that these helix-helix interactions are actually mediated in part by the hydrophobic acyl chains of the lipids.…”