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The membrane-bound structure and topology of a human ?-defensin indicate a dimer pore mechanism for membrane disruption.

ABSTRACT: 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 ?-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 ?2 and ?3, whose side chains exhibit immobilization and conformational perturbation upon membrane binding. At high protein/lipid ratios, rapid (1)H 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?-lipid (31)P 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 (19)F 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 dimer 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 ?-hairpin antimicrobial peptide. These results provide the first glimpse into the membrane-bound structure and mechanism of action of human ?-defensins.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC2992833 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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The membrane-bound structure and topology of a human α-defensin indicate a dimer pore mechanism for membrane disruption.

Zhang Yuan Y Lu Wuyuan W Hong Mei M

Biochemistry 20101026 45

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 α-defensin, HNP-1, in DMPC/DMPG bilayers. Two-dimensional ...[more]

PMID: 20961099

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Project description:Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.

Project description:Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I(2) synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A (1 A=0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare site-specific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the accessibility of the substrate access channel opening in transiently transfected COS-1 cells expressing recombinant human PGIS, and in spontaneously transformed human endothelial cell line ECV cells expressing endogenous human PGIS. Immunofluorescence staining was performed for cells selectively permeabilized with streptolysin O and for cells whose membranes were permeabilized with detergent. Antibodies to peptides in the immediate vicinity of the substrate channel (P66-75 and P95-116) bound to their targets only after general permeabilization with Triton X-100. In contrast, the two antibodies to peptides further from the channel opening (P366-382 and P472-482) bound to their targets even in cells with intact ER membranes. These observations support our topology model in which the PGIS substrate access channel opening is positioned close to the ER membrane.

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The membrane-bound structure and topology of a human ?-defensin indicate a dimer pore mechanism for membrane disruption.

Publications

The membrane-bound structure and topology of a human α-defensin indicate a dimer pore mechanism for membrane disruption.

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