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.
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 mum. Circular dichroism spectrometry revealed that the S3 peptide undergoes conformational change in the presence of a disulfide bridge, transitioning from a random coil to beta-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.
Li
Peng
P
Departments of Biological Sciences, Chemistry, and Microbiology, National University of Singapore, 117543 Singapore.
Wohland
Thorsten
T
Ho
Bow
B
Ding
Jeak Ling
JL
D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008823:Micelles; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*
D004220:Disulfides; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D008823:Micelles; D009363:Neoplasm Proteins; D010446:Peptide Fragments; C116881:SRPX2 protein, human