Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1)

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Medite rranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synth esis and an easy way to improve the yield of the four native disulfide bond s. Synthetic and native MGD-1 display similar antibacterial activity, sugge sting that the hydroxylation of Trp28 observed in native MGD-1 is not invol ved in the antimicrobial effect. The three-dimensional solution structure o f MGD-1 has been established using H-1 NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta -strands (Arg20-Cys25 and Cys3 3-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabili zed alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys 10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 dis ulfide bond which is solvent-exposed, the three others belong to the partic ularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positiv e activity, suggesting that the fourth disulfide bond of MGD-1 is not essen tial for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typi cal distribution of positively charged and hydrophobic side chains. The pos itively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that t he rather well conserved location of certain positively charged residues an d of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.