SMAP‐29 has two LPS‐binding sites and a central hinge

Abstract

The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8-17 were helical, residues 18-19 formed a hinge, and residues 20-28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC 50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC 50 of the peptide (2.6 lM) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N-and C-terminal LPS-binding sites of the same peptide molecule). , an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N-and C-terminal LPS-binding domains, had an EC 50 of 2.5 lM, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.

Peptide interactions with bacterial lipopolysaccharides

“…As discussed below, it has been widely noted in literature that many 7 structural characteristics are similar for AMPs and anti-endotoxic peptides, e.g., positive charge and amphiphilicity, and many anti-endotoxic peptides are indeed also AMPs. This was illustrated, e.g., by Tack et al who investigated variants of the antimicrobial peptide SMAP-29, and found a good correlation between minimal affective concentrations (MECs) for bacteria killing, on one hand, and LPS binding affinity, on the other (Figure 3) (15). Although many anti-endotoxic peptides are also AMPs, the reverse does not necessarily hold, suggesting that the membrane binding characteristics of AMPs, or underlying characteristics resulting in membrane binding and rupture, constitute a necessary, but not sufficient, requirement for anti-endotoxic effects.…”

Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish

“…In addition to the direct antimicrobial effects, mammalian CAMPs possess other modulatory activities, such as LPS-neutralizing (20,21) and IL-8-stimulating activities (22,23,28,38,39). Furthermore, the LPS-binding activities are often positively correlated with their antibacterial activities (21).…”

“…Binding of peptides to LPS was expressed as a percentage of absorbance developed by 0.1 mg peptide. Additionally, the LPS-binding isotherms of trout CAMPs and their truncated variants were quantified by the kinetic chromogenic Limulus amebocyte lysate assay (QCL-1000 kit; Lonza) as previously described (21,37).…”

“…These CAMPs exhibit a broad spectrum of antimicrobial activities against bacteria, viruses, and fungi (2,6,18). In addition to their direct antimicrobial effects, mammalian CAMPs possess other modulatory activities, such as LPS neutralizing (20,21) and cytokine stimulating (22,23).…”

Analysis of the solution structure of the human antibiotic peptide dermcidin and its interaction with phospholipid vesicles

“…The structure of DCD includes a highly flexible N-terminal helical region and a relatively ordered C-terminal helical region. A structural homology search based on the helix-hingehelix motif suggests DCD resembles a variety of antimicrobial peptides, including cecropin A, gaegurin 4 (GGN4) and SMAP-29 (23,26,27). DCD-1L shows particular similarity to SMAP-29, an antimicrobial peptide from sheep (27) that presents a flexible N-terminal region (residues 1 to 6), a helical region (residues 8 to 17), a C-terminal helical region (residues 20 to 28) and a hinge segment (Gly18 and Pro19).…”