Solution Structures of Human LL-37 Fragments and NMR-Based Identification of a Minimal Membrane-Targeting Antimicrobial and Anticancer Region

Abstract

To understand the structure and activity relationship of human LL-37, a series of peptide fragments was designed. The N-terminal fragment, LL-37(1-12), was not active, while the C-terminal fragment, LL-37(13-37), killed Escherichia coli, as well as drug-sensitive and drug-resistant cancer cells. A 13-residue core antibacterial and anticancer peptide, corresponding to residues 17-29 of LL-37, was identified based on total correlated spectroscopy by trimming nonessential regions (TOCSY-trim). Because LL-37 acts on bacterial membranes, three-dimensional structures of its fragments were determined in micelles by NMR, including structural refinement by natural abundance 15N and 13C chemical shifts. Aromatic-aromatic interactions in the N-terminal fragment were proposed to be essential for LL-37 aggregation. The LL-37 core peptide adopts a similar structure in the micelles of SDS or dioctanoyl phosphatidylglycerol. This structure is retained in the C-terminal fragment LL-37(13-37) and very likely in intact LL-37 based on peptide-aided signal assignments. The higher antibacterial activity of the LL-37 core peptide than aurein 1.2 was attributed to additional cationic residues. To achieve selective membrane targeting, D-amino acids were incorporated into LL-37(17-32). While the D-peptide showed similar antibacterial activity to the L-diastereomer, it lost toxicity to human cells. Structural analysis revealed hydrophobic defects in the new amphipathic structure of the D-peptide, leading to a much shorter retention time on a reversed-phase HPLC column. It is proposed that hydrophobic defects as a result of incoherent hydrophobic packing provide a structural basis for the improvement in cell selectivity of the LL-37 fragment.

Improvement of the Efficacy of Linear Undecapeptides against Plant-Pathogenic Bacteria by Incorporation of d -Amino Acids

“…D-Amino acid incorporation has been previously applied to other antimicrobial peptides, such as pardaxin, melittin, or magainin, leading to D-analogues with similar antimicrobial activities, lower hemolytic activity levels, and higher stability levels against protease degradation (11,28). In the present study, the antibacterial activity of the BP100 isomers depended on the pathogen, the number of D-amino acids introduced, and the residue that was replaced.…”

“…In particular, incorporation of D-amino acids is an approach used to protect peptides against enzymatic hydrolysis, since only a few enzymes are known to digest amide bonds involving D-configuration (32). This strategy has been used to improve the biological activity profiles of synthetic antimicrobial peptides, not only increasing the resistance to proteolytic enzymes but also reducing the hemolytic activity while maintaining the antimicrobial activity (23,28,42,44,47,48,54).…”

d -Alanine Modification of a Protease-Susceptible Outer Membrane Component by the Bordetella pertussis dra Locus Promotes Resistance to Antimicrobial Peptides and Polymorphonuclear Leukocyte-Mediated Killing

“…The SPLUNC1 (short palate, lung, nasal epithelium clone 1) protein is a crucial component of innate immunity and is highly expressed in the oral cavity and respiratory tract of humans and many other mammals (41)(42)(43). Human CAP18/LL-37 (hCAP18/LL-37) is a prototype member of the human cathelicidin family and is produced by neutrophils, macrophages, and various epithelial cells (44).…”

Decoding the Functional Roles of Cationic Side Chains of the Major Antimicrobial Region of Human Cathelicidin LL-37

“…As a complementary approach to understanding the structureactivity relationship of LL-37, several active fragments were identified using synthetic peptide libraries, in silico design, or protease digestion (5,31,37,50,51). Among these fragments, GF-17, corresponding to residues 17 to 32 of LL-37 ( Fig.…”