We previously reported the de novo design of an amphiphilic peptide @YGG~IEKKIEA! 4 # that forms a native-like, parallel triple-stranded coiled coil. Starting from this peptide, we sought to regulate the assembly of the peptide by a metal ion. The replacement of the Ile18 and Ile22 residues with Ala and Cys residues, respectively, in the hydrophobic positions disrupted of the triple-stranded a-helix structure. The addition of Cd~II!, however, resulted in the reconstitution of the triple-stranded a-helix bundle, as revealed by circular dichroism~CD! spectroscopy and sedimentation equilibrium analysis. By titration with metal ions and monitoring the change in the intensity of the CD spectra at 222 nm, the dissociation constant K d was determined to be 1.5 6 0.8 mM for Cd~II!. The triple-stranded complex formed by the 113 Cd~II! ion showed a single 113 Cd NMR resonance at 572 ppm whose chemical shift was not affected by the presence of Cl Ϫ ions. The 113 Cd NMR resonance was connected with the bH protons of the cysteine residue by 1 H-113 Cd heteronuclear multiple quantum correlation spectroscopy. These NMR results indicate that the three cysteine residues are coordinated to the cadmium ion in a trigonal-planar complex. Hg~II! also induced the assembly of the peptide into a triple-stranded a-helical bundle below the Hg~II!0peptide ratio of 103. With excess Hg~II!, however, the a-helicity of the peptide was decreased, with the change of the Hg~II! coordination state from three to two. Combining this construct with other functional domains should facilitate the production of artificial proteins with functions controlled by metal ions.Keywords: coiled coil; de novo design; folding; helical structures; metalloproteins Studies on de novo designed proteins involve the construction of proteins with unique tertiary structures and the creation of new functional proteins. The coiled coil structure is often observed in natural proteins for the intermolecular assemblies of the functional domains. This motif, due to its structural simplicity and biomolecular significance, has been the subject of extensive analyses to understand the principles of de novo design, as well as protein folding and stability~Lau et al., 1984;O'Neil & DeGrado, 1990;Harbury et al., 1993!. Furthermore, the designed coiled coils can be fused to various functional peptides or domains of natural proteins for biological and medical applications~Pack & Plückthun, 1992; Hodges, 1996; Terskikh et al., 1997!.The designed coiled coil, which drastically changes its conformation depending on external stimuli, should be useful to control the associations and the functions of domains attached to the peptide. Among the various external stimuli, metal binding has been studied extensively, and the factors required for metal binding are well understood. A variety of metal binding sites have been designed, but they are on the surfaces of preformed artificial supramolecules, such as an a-helical bundle protein~Handel & Regan & Clarke, 1990;Regan, 1995;Dieckmann et...
The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration-and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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