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J. Biol. Chem., Vol. 279, Issue 47, 48671-48679, November 19, 2004

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Structure-Activity Relationships in Defensin Dimers

A NOVEL LINK BETWEEN -DEFENSIN TERTIARY STRUCTURE AND ANTIMICROBIAL ACTIVITY^*

Dominic J. Campopiano, David J. Clarke, Nick C. Polfer, Perdita E. Barran¶, Ross J. Langley||, John R. W. Govan||, Alison Maxwell**, and Julia R. Dorin**

From the School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, the ^||Department of Medical Microbiology, University Medical School, University of Edinburgh, Edinburgh EH8 9AG, and ^**Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a -defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mM NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have -defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.

Received for publication, April 27, 2004 , and in revised form, August 6, 2004.

^* This work was supported by the Biotechnology and Biological Sciences Research Council, the Engineering and Physical Sciences Research Council, the Cystic Fibrosis Trust, the Medical Research Council UK, Scottish Enterprise, and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed. Tel.: 44-131-650-4712; Fax: 44-131-650-4743; Dominic.Campopiano{at}ed.ac.uk. ¶ To whom correspondence may be addressed. Tel.: 44-131-650-7533; Fax: 44-131-650-7533; E-mail: Perdita.Barran{at}ed.ac.uk.

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