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

Abstract

Dermcidin is a human antibiotic peptide that is secreted by the sweat glands and has no homology to other known antimicrobial peptides. As an initial step toward understanding dermcidin's mode of action at bacterial membranes, we used homonuclear and heteronuclear NMR to determine the conformation of the peptide in 50% trifluoroethanol solution. We found that dermcidin adopts a flexible amphipathic α-helical structure with a helix-hinge-helix motif, which is a common molecular fold among antimicrobial peptides. Spin-down assays of dermcidin and several related peptides revealed that the affinity with which dermcidin binds to bacterial-mimetic membranes is primarily dependent on its amphipathic α-helical structure and its length (＞30 residues); its negative net charge and acidic pI have little effect on binding. These findings suggest that the mode of action of dermcidin is similar to that of other membrane-targeting antimicrobial peptides, though the details of its antimicrobial action remain to be determined [BMB reports 2010; 43(5): 362-368] INTRODUCTIONAntimicrobial peptides are produced in a wide range of animals for innate host defense and show a broad spectrum of antimicrobial activity (1-4). Despite the remarkable diversity of their amino acid compositions, antimicrobial peptides show several common features, including the presence of multiple basic amino acids and amphipathic structures with clusters of hydrophobic and hydrophilic amino acids (5-8). Recent studies of the modes of action of various antimicrobial peptides have revealed that their cationic nature contributes to their initial binding to negatively charged bacterial membranes through electrostatic interaction, while their amphipathic structures enhance peptide-lipid interactions at the water-bilayer interface, ultimately leading to cell death via pore formation or membrane disintergration (9-14).Dermcidin (DCD) is an antimicrobial peptide found in human sweat. It is produced through proteolytic processing of a 110-amino acid precursor protein that has no homology to other known antimicrobial peptides (15, 16). The processed peptide, DCD-1, has 47 amino acids and shows a broad spectrum of antimicrobial activity against a variety of pathogenic microorganisms (15,17,18). Using immune-EM, it was recently shown that the antimicrobial activity of DCD-1 originates with its binding to the bacterial membrane, and that it effectively kills Staphylococcus epidermidis (18).DCD-1L, which is produced by adding a leucine residue to the C-terminus of DCD-1, shows stronger antimicrobial activity than the parent peptide (15). It is especially noteworthy that DCD-1L exhibits activity against drug-resistant S. aureus, as well as other Gram-positive and Gram-negative bacterial strains (17). In the present study, we used nuclear magnetic resonance (NMR) spectroscopy to determine, for the first time, the solution structure of DCD-1L in a bacterial membrane-mimetic environment. In addition, to investigate the interaction between DCD-1L and bacterial membranes, we sy...

Human Antimicrobial Peptides and Proteins

“…It should be pointed out that the 3D structure of DCD-1L was also determined previously by 3D NMR spectroscopy using a 15 N-labeled peptide in a 50% trifluoenthanol (TFE) solution. Four helical regions were identified (α1: 5–7; α2: 10–12; α3: 26–33; and α4: 36–45) [217]. In this case, TFE might have disrupted the oligomeric structure of dermcidin.…”

The Multiple Facets of Dermcidin in Cell Survival and Host Defense

“…b NMR secondary structure of DCD-1L in 50% TFE is shown (taken from http://www.rcsb.org/pdb/explore.do? structureId=2ksg; see also Jung et al [79]). c Schematic view of human skin with the different epidermal layers and an eccrine sweat gland secreting sweat (brown dots at the skin surface).…”

“…DCD-1L easily self-associates to form higher-order oligomers and has an anomalous electrophoretic mobility in the presence of SDS molecules [35,77]. Solution-NMR analyses in 50% TFE indicate that the DCD-1L topology consists of four α-helices and is highly flexible, especially the N-terminus of DCD-1L (amino acid position 1–23) [79] (fig. 3b) .…”

Structural basis for the bacterial membrane insertion of dermcidin peptide, DCD-1L

“…In the current study, we determined solution NMR structures of DCD-1L in 60 mM sodium dodecyl sulfate (SDS) micelles without Zn 2+ , and found a much higher helical content than that found by Jung and colleagues in 50% TFE 12 . The structure of DCD-1L in the SDS micelle showed an “L-shaped” molecule with three α-helices connected by flexible turns.…”

“…The N-terminal half (S2–K23) of DCD-1L is more unstructured as compared with the C-terminal portion (D24–L48). Furthermore, the membrane affinity is found to depend on the amphipathic structure, helical content and length of the DCD peptide, rather than its negative net charge or isoelectric point 12 .…”