Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 n
m concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.LPS
2 or endotoxin, a major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is critically involved in health and diseases of humans (
1,
2). LPS is essential for bacterial survival through establishing an efficient permeability barrier against a variety of antimicrobial compounds including hydrophobic antibiotics, detergents, host-defense proteins, and antimicrobial peptides (
3,
4). Several studies have demonstrated that LPS catalyzes folding of outer membrane proteins as a chaperone (
5–
7).LPS, a potent inducer of innate immune systems, hence called endotoxin, is primarily responsible for lethality in sepsis and septic shock syndromes associated with serious Gram-negative infections (
8–
10). Circulating LPS in bloodstream is intercepted by the phagocytic cells of the innate immune system. Once induced by LPS, these phagocytes produce proinflammatory cytokines,
e.g. tumor necrosis factor-α, interleukin-6, and interleukin-1β, through the activation of a Toll-like pattern recognition receptor (
11,
12). The release of cytokines in response to microbial invasion is a natural function of the innate immunity. However, an uncontrolled and overwhelming production of these cytokines may cause “endotoxic shock” or septic shock, typified by endothelial tissue damage, loss of vascular tone, coagulopathy, and multiple organ failure, often resulting in death (
9,
10). Sepsis is the major cause of mortality in the intensive care unit, accounting for 200,000 deaths every year in the United States alone (
13). It was demonstrated that release of LPS from antibiotic-treated Gram-negative bacteria can indeed enhance sepsis (
14). Therefore, an effective antibiotic should not only exert antibacterial activities but also have the ability to sequester LPS and ameliorate its toxicity. Therefore, an amalgamated property of LPS-neutralizing and antimicrobial activity would be highly desirable for antimicrobial agents. Polymyxin B is a prototypical antimicrobial and antiendotoxic antibiotic; however, its neurotoxicity and nephrotoxicity limit its application to topical use (
15). The increasing emergence of bacterial strains that are resistant to conventional antibiotics has initiated vital structure/function studies of membrane-perturbing cationic antimicrobial peptides (
16–
20). More recent studies have been conducted to understand interactions between antimicrobial peptides with LPS to gain insights into the mechanism of outer membrane perturbation, antibacterial activities, and LPS neutralization (
21–
26). These studies have delineated the role of amino acid sequence properties, LPS-peptide interactions by biophysical methods, and global structural parameters, obtained by CD and FTIR.Designing synthetic peptides and elucidation of three-dimensional structures in complex with LPS would be useful for the purpose of rational development of non-toxic antisepsis and antimicrobial therapeutics. Such studies will also be potentially instructive in establishing rules by which folded structures can be stabilized on the LPS surface. Extensive work in the field of peptide design primarily focuses on mimicking secondary structures and tertiary folds of proteins. Usually, short linear peptides are often structurally flexible; however, the functions of these peptides are highly dependent on their ability to adopt folded structures upon complex formation with their cognate receptors. In this regard, designed peptides that would yield high resolution structures in complex with LPS have not been well pursued. LPS, being a negatively charged amphiphilic molecule, interacts with naturally occurring peptides or protein fragments containing basic/polar and hydrophobic amino acids, although there are considerable variations in lengths, sequences, and amino acid compositions among these peptides (
27,
28).Here, we have determined the three-dimensional structures of a series of 12-residue peptides in the context of LPS. To the best of our knowledge, these results show, for the first time, that atomic resolution structures of designed peptides obtained in LPS could be correlated with their antiendotoxic activities. Furthermore, the LPS-induced structures of active, inactive, and short peptide motif, presented here, may provide building blocks for the designing novel proteins for the outer membrane.
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