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J. Biol. Chem., Vol. 279, Issue 48, 50150-50156, November 26, 2004

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Perturbation of Lipopolysaccharide (LPS) Micelles by Sushi 3 (S3) Antimicrobial Peptide

THE IMPORTANCE OF AN INTERMOLECULAR DISULFIDE BOND IN S3 DIMER FOR BINDING, DISRUPTION, AND NEUTRALIZATION OF LPS^*

Peng Li, Thorsten Wohland¶, Bow Ho||, and Jeak Ling Ding**

From the Departments of Biological Sciences, ^¶Chemistry, and ^||Microbiology, National University of Singapore, 117543 Singapore

S3 peptide, derived from the Sushi 3 domain of Factor C, which is the lipopolysaccharide (LPS)-sensitive serine protease of the horseshoe crab coagulation cascade, was shown previously to harbor antimicrobial activity against Gram-negative bacteria. However, the mechanism of action remains poorly understood at the molecular level. Here we demonstrate that the intermolecular disulfide bonding of S3 resulting in S3 dimers is indispensable for its interaction with LPS. The binding properties of the S3 monomer and dimer to LPS were analyzed by several approaches including enzyme-linked immunosorbent assay (ELISA)-based assay, surface plasmon resonance, and fluorescence correlation spectroscopy (FCS). It is evident that the S3 dimer exhibits stronger binding to LPS, demonstrating 50% LPS-neutralizing capability at a concentration of 1 µM. Circular dichroism spectrometry revealed that the S3 peptide undergoes conformational change in the presence of a disulfide bridge, transitioning from a random coil to -sheet structure. Using a fluorescence correlation spectroscopy monitoring system, we describe a novel approach for examining the mechanism of peptide interaction with LPS in the native environment. The strategy shows that intermolecular disulfide bonding of S3 into dimers plays a critical role in its propensity to disrupt LPS micelles and consequently neutralize LPS activity. S3 dimers display detergent-like properties in disrupting LPS micelles. Considering intermolecular disulfide bonds as an important parameter in the structure-activity relationship, this insight provides clues for the future design of improved LPS-binding and -neutralizing peptides.

Received for publication, May 19, 2004 , and in revised form, August 24, 2004.

^* This work was supported by the Agency for Science and Technology Research (A* STAR) of Singapore. 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.

Recipient of a Research Scholarship from the National University of Singapore.

^** To whom correspondence should be addressed: Dept. of Biological Sciences, National University of Singapore, 14, Science Dr. 4, 117543 Singapore. Tel.: 65-6874-2776; Fax: 65-6779-2486; E-mail: dbsdjl{at}nus.edu.sg; World Wide Web: www.dbs.nus.edu.sg/Staff/ding.html.

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