ABSTRACT
α-Hordothionin (αHTH) belongs to thionins, the plant antimicrobial peptides with membrane-permeabilizing activity which is associated with broad-range antimicrobial activity. Experimental data have revealed a phospholipid-binding site and indicated formation of ion channels as well as membrane disruption activity of thionin. However, the mechanism of membrane permeabilization by thionin remained unknown. Here it is shown that thionin is a small water-selective channel. Unbiased high-precision molecular modeling revealed formation of a water-selective pore running through the αHTH double α-helix core when the peptide interacted with anions. Anion-induced unfolding of the C-end of the α2-helix opened a pore mouth. The pore started at the α2 C-end between the hydrophilic and the hydrophobic regions of the peptide surface and ended in the middle of the unique hydrophobic region at the C-end of the α1-helix. Highly conserved residues including cysteines and tyrosine lined the pore walls. A large positive electrostatic potential accumulated inside the pore. The narrow pore was, nonetheless, sufficient to accommodate at least one water molecule along the channel except for two constriction sites. Both constriction sites were formed by residues participating in the phospholipid-binding site. The channel properties resembled that of aquaporins with two selectivity filters, one at the entrance, inside the α2 C-end cavity, and a second in the middle of the channel. It is proposed that the αHTH water channel delivers water molecules to the bilayer center that leads to local membrane disruption. The proposed mechanism of membrane permeabilization by thionins explains seemingly controversial experimental data.
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Amino Acid Sequence , Antimicrobial Cationic Peptides/genetics , Binding Sites , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Plant Proteins/genetics , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Water/chemistry , Water/metabolismABSTRACT
Computational analysis of two membrane-permeabilizing peptides, barley alpha-hordothionin and wheat beta-purothionin, revealed that anions can trigger dynamic and structural changes in the thionin antiparallel double alpha-helix core. Analysis of the molecular dynamics simulations demonstrated that anions induced unfolding of the alpha2 and alpha1 helices at the carboxyl ends which are located on the opposite ends of the alpha-helix core. An internalized water molecule was observed inside the unfolded alpha2 C-end. Strong interactions of anions with the R30 regulating network or simultaneous interactions of anions with the phospholipid-binding site and the R30 hydrogen bonding network triggered unfolding of the alpha2 C-end. An increase of anion density for two residues of the phospholipid-binding site (K1, R17, and Q22) or R17 and R19 and a preceding unfolding of the alpha2 C-end were necessary for unfolding of the alpha1 C-end. Anions interacted primarily with residues of the phospholipid-binding site and the R30 network while the alpha1/alpha2 hydrophobic region was void of anions. However, during strong interactions of anions with the R30 network and phospholipid-binding site, the alpha1/alpha2 hydrophobic region attracted anions which interacted with conserved residues of the alpha1 C-end. Analysis of anion-induced rearrangements pointed to auxiliary residues of the R30 network and the phospholipid-binding site. Induction of conformational changes on the opposite ends of the alpha-helix core by interactions of anions with the phospholipid-binding site may be relevant to a mechanism of membrane-permeabilizing activity.
