Antimicrobial peptides (AMPs) are present in all species investigated to date. They form an important part of innate immunity, protecting the organism from infection by directly killing invading bacteria. Since pathogenic microorganism show an increasing tendency to be immune against common antibiotics, AMPs carry remarkable pharmaceutical promise as next-generation antibiotics.
The electron micrographs below show how a bacterium is affected by LL-37, a human antimicrobial peptide. The bacterium dies if a threshold called 'minimum inhibitory concentration' (MIC) is reached. Even at concentrations below the MIC, the bacterium shows visible damage.
The exact mechanism by which AMPs kill microorganisms is still under debate. The cartoon below summarizes aspects known for the action of AMPs. In solution (top row of the cartoon), AMPs may be found in unordered conformation as well as in defined secondary structure. In some cases, AMPs aggregate to form oligomers in solution.
Binding to microbial membranes is the first step for AMP to kill bacteria. Currently it is not known which conformation in solution leads to membrane binding. After binding, all AMP display defined secondary structure. Regularly, membrane-bound AMP are observed to form dimers or higher oligomers.
After binding, AMP make bacterial membranes permeable for ions and other cellular content, thus harming or killing the bacterium. Two molecular models to explain the membrane permeability caused by AMP are illustrated on the bottom of the cartoon. AMPs may act as detergent, seriously perturbing the structural integrity of the membrane ('carpet model', left). Another possible way of action may be the formation of actual protein-lined membranes pores ('barrel stave model', right). Our lab has studied a large variety of antimicrobial peptides with all sorts of secondary structure. Currently, we are focusing on two human AMPs, HBD and LL-37.
Human AMPs: Dozens of AMPs are found in the human organism. The alpha- and beta-defensins form the largest group. The cathelicidin group, found in all species investigated, has only a single representative in human, called LL-37. Both LL-37 and beta-defensins are under investigation in our lab.
Human beta-defensin 3 (HBD-3) is an antimicrobial peptide with a complex molecular structure, shown left in the figure below. The structure consists of a three-stranded b-sheet, an a-helical portion and several disordered loops. Also shown are the three disulfide bridges that stabilize the molecular structure. HBD-3 is known to form dimers in solution, a model is shown on the right of the figure. Currently we investigate several mutants and fragments of HBD-3 in order to identify minimal structural motifs needed to be active against bacteria.
The human cathelicidin LL-37 is an alpha-helical amphipathic peptide. The figure below shows an electrostatic surface plot and a cartoon representation of LL-37, stressing the amphipathic nature of the peptide. The observed topology with a hydrophilic surface on the top and a hydrophobic surface on the bottom of the peptide is a common feature of all antimicrobial peptides. It allows for membrane binding with the hydrophobic face of the AMP inserting into the hydrophobic core of the lipid bilayer. Note that this amphipathic topology is disturbed at the N-terminus of LL-37. This altered topology may be necessary for LL-37 to perform the complex signaling function which it serves in addition to its antimicrobial functions.
We have investigated LL-37's structure in lipid bilayers using 13C- and 15N-labeled LL-37. The 15N-NMR spectra of aligned and unaligned samples (bottom left) show that LL-37 aligns with the amphipathic bilayer interface. In addition, 2H- and 31P-NMR of the bilayer lipids revealed a severe distortion of the hydrophobic bilayer core in the presence of LL-37. The cartoon on the right summarizes the findings of bilayer perturbation and surface alignment of the peptide.
Currently we hope to extend these studies by the use of uniformly 15N-labeled LL-37, and by experiments designed to detect and characterize a probable oligomerization of LL-37.
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