The Acyl Group as the Central Element of the Structural Organization of Antimicrobial Lipopeptide Bos ˇ tjan Japelj, † Mateja Zorko, † Andreja Majerle, † Primoz ˇ Pristovs ˇ ek, † Susana Sanchez-Gomez, ‡ Guillermo Martinez de Tejada, ‡ Ignacio Moriyon, ‡ Sylvie E. Blondelle, § Klaus Brandenburg, | Jo ¨rg Andra ¨, | Karl Lohner, ⊥ and Roman Jerala* ,† Laboratory of Biotechnology, National Institute of Chemistry, HajdrihoVa 19, 1000 Ljubljana, SloVenia, Department of Microbiology and Parasitology, UniVersity of NaVarra, C/ Irunlarrea n° 1, 31008 Pamplona, Spain, Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121, DiVision of Biophysics, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 10, D-23845 Borstel, Germany, Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Schmiedlstrasse 6, A-8042 Graz, Austria Received October 17, 2006; E-mail: roman.jerala@ki.si Incorporation of non-natural amino acids or non-amino acid groups into peptides, such as -amino acids, carbohydrates, nucleotides, or fatty acids extends their spectrum of activities as well as the accessible conformational space. In most of the determined structures of such hybrids, the peptide and non-peptide segment are segregated into two separate domains. Here we report on the solution structure of an antimicrobial lipopeptide, which shows that the acyl chain is tightly intertwined with the amino- acid residues. An acyl chain is present on several natural peptide antibiotics produced by nonribosomal synthesis. The acyl chain of polymyxin B, the archetypal lipopolysaccharide (LPS) neutralizing lipopeptide is essential for its antimicrobial activity. 1 Following the paradigm of polymyxin B, conjugation of cationic antimicrobial peptides with fatty acid chains improved their antimicrobial activity against Gram-negative and Gram-positive bacteria but also neu- tralization of LPS. 2-4 An ideal antimicrobial agent should display not only antimicrobial activity but also the ability to bind to and neutralize bacterial endotoxin in order to prevent septic (or endotoxic) shock, which accounts for more than 200 000 deaths in the U.S. every year. 5 We have shown previously that the lipid environment signifi- cantly affects the conformation of LF11 (FQWQRNIRKVR-NH 2 ), a short antimicrobial peptide derived from human lactoferrin. 6 The acyl chain has been generally treated as a simple hydrophobic “appendix” that exerts no significant structural effects in the peptide segment, 7-9 and the improvement of antimicrobial and endotoxin- neutralizing activities has been attributed to a simple increase of bulk hydrophobicity. 2,10-15 In this paper we report on the effect of N-terminal acylation of this peptide (C12LF11 or N-lauryl-LF11, CH 3 -(CH 2 ) 10 -CO-NH-FQWQRNIRKVR-NH 2 ) on the structural organization of the peptide in the membrane mimetic environment, which is reflected in its antimicrobial activity modulated also by bacterial LPS. In order to discern the role of LPS on the antimicrobial activity of C12LF11, we used a collection of six isogenic Salmonella minnesota strains carrying well-defined muta- tions in genes involved in the biosynthesis of LPS. These mutants make rough LPS differing in their carbohydrate core size. Thereby the antimicrobial activity of C12LF11 was inversely related to the carbohydrate core size. Further, in all strains the lipopeptide activity was 4-16 fold better than the nonacylated peptide (Table S2). This is in agreement with the proposed effect of the negatively charged carbohydrate groups of LPS, which can act as a “sponge” to prevent binding of cationic peptides to the membrane inserted lipid A group. 16 The C12LF11 peptide also significantly decreased the mortality of mice inoculated with P. aeruginosa LPS (to be described elsewhere). The increased affinity of the lipopeptide for LPS results in the elimination of the transferred NOE effect that would allow structure determination of the complex as previously for the parent peptide LF11, 6 but may facilitate the transport across the outer membrane as the self-promoted cell uptake of polymyxin B. 17 Nevertheless, CD spectra show that LPS has a larger structuring effect on C12LF11 than on the nonacylated peptide (Figure S3, Supporting Information). Furthermore, in aqueous environment no persistent structure of C12LF11 was observed, while a significant effect of acylation on the peptide’s conformation in the membrane- mimetic environment is seen, which is more prominent in DPC than in SDS (Figure S3). Structure of C12LF11 in Dodecylphosphocholine (DPC) Micelles. 2D NOESY NMR spectra of C12LF11 in the presence of DPC (single chain phosphocholine) micelles revealed a large number of cross-peaks that could be unanimously assigned because of the high dispersion of the signals, notably including those between the peptide and the acyl chain (Figure 1). The hydrogen atoms attached to the R, , and γ C atoms of the acyl chain showed NOEs with Phe 1 , Gln 2 , Trp 3 , and Gln 4 . The refined C12LF11 structure in DPC micelles was well-defined with a backbone rmsd of 0.44 Å for residues 2-10 (Figure S1). It contained two short R-helical stretches (Phe 1 , to Arg 5 and Asn 6 to Arg 11 ) tilted at an angle of 90°. Arg 5 , Arg 8 , Lys 9 , and Arg 11 formed an almost linear cluster of positively charged residues, whereas the acyl chain atoms, Phe 1 , Gln 2 , Trp 3 , Ile 7 , and Val 10 were grouped in a hydrophobic patch, which gave the molecule a distinct amphi- pathic character. Structure of C12LF11 in Sodium Dodecylsulphate (SDS) Micelles. The R and  protons in the acyl chain showed NOEs with Phe 1 -H δ and aromatic protons of Trp. 3 The refined C12LF11 structure in SDS was less defined than in zwitterionic DPC (backbone rmsd for residues 2-10 1.72 Å (Figure S2)) in agreement with CD data. The residues Asn 6 , Ile 7 , Arg 8 , Lys 9 , and Val 10 were organized in an extended conformation, with the side chains of the four basic residues Arg 5 , Arg 8 , Lys 9 , and Arg 11 forming a concavely shaped surface. The hydrophobic core (Phe 1 , Trp 3 , Ile 7 , and Val 10 ) was significantly better defined (heavy atom rmsd 1.64 ( 0.71 Å) than the rest of the peptide; its side chains were oriented opposite † National Institute of Chemistry. ‡ University of Navarra. § Torrey Pines Institute for Molecular Studies. | Leibniz Center for Medicine and Biosciences. ⊥ Austrian Academy of Sciences. Published on Web 01/12/2007 1022 9 J. AM. CHEM. SOC. 2007, 129, 1022-1023 10.1021/ja067419v CCC: $37.00 © 2007 American Chemical Society