The Membrane-Bound Structure and Topology of a Human α-Defensin Indicate a Dimer Pore Mechanism for Membrane Disruption

Defensins are cationic and disulfide-bonded host defense proteins of many animals that target microbial cell membranes. Elucidating the three-dimensional structure, dynamics, and topology of these proteins in phospholipid bilayers is important for understanding their mechanisms of action. Using solid-state nuclear magnetic resonance spectroscopy, we have now determined the conformation, dynamics, oligomeric state, and topology of a human alpha-defensin, HNP-1, in DMPC/DMPG bilayers. Two-dimensional correlation spectra show that membrane-bound HNP-1 exhibits a conformation similar to that of the water-soluble state, except for the turn connecting strands beta 2 and beta 3, whose side chains exhibit immobilization and conformational perturbation upon membrane binding. At high protein/lipid ratios, rapid H-1 spin diffusion from the lipid chains to the protein was observed, indicating that HNP-1 was well inserted into the hydrocarbon core of the bilayer. Arg C zeta-lipid P-31 distances indicate that only one of the four Arg residues forms tight hydrogen-bonded guanidinium-phosphate complexes. The protein is predominantly dimerized at high protein/lipid molar ratios, as shown by F-19 spin diffusion experiments. The presence of a small fraction of monomers and the shallower insertion at lower protein concentrations suggest that HNP-1 adopts concentration-dependent oligomerization and membrane-bound structure. These data strongly support a dimer pore topology of HNP-1 in which the polar top of the dither lines an aqueous pore while the hydrophobic bottom faces the lipid chains. In this structure, R25 lies closest to the membrane surface among the four Arg residues. The pore does not have a high degree of lipid disorder, in contrast to the toroidal pores formed by protegrin-1, a two-stranded beta-hairpin antimicrobial peptide. These results provide the first glimpse into the membrane-bound structure and mechanism of action of human alpha-defensins.