High-quality 3D structures shine light on antibacterial, anti-biofilm and antiviral activities of human cathelicidin LL-37 and its fragments

Abstract

Host defense antimicrobial peptides are key components of human innate immunity that plays an indispensible role in human health. While there are multiple copies of cathelicidin genes in horses, cattle, pigs, and sheep, only one cathelicidin gene is found in humans. Interestingly, this single cathelicidin gene can be processed into different forms of antimicrobial peptides. LL-37, the most commonly studied form, is not only antimicrobial but also possesses other functional roles such as chemotaxis, apoptosis, wound healing, immune modulation, and cancer metastasis. This article reviews recent advances made in structural and biophysical studies of human LL-37 and its fragments, which serve as a basis to understand their antibacterial, anti-biofilm and antiviral activities. High-quality structures were made possible by using improved 2D NMR methods for peptide fragments and 3D NMR spectroscopy for intact LL-37. The two hydrophobic domains in the long amphipathic helix (residues 2-31) of LL-37 separated by a hydrophilic residue serine 9 explain its cooperative binding to bacterial lipopolysaccharides (LPS). Both aromatic rings (F5, F6, F17, and F27) and interfacial basic amino acids of LL-37 directly interact with anionic phosphatidylglycerols (PG). Although the peptide sequences reported in the literature vary slightly, there is a consensus that the central helix of LL-37 is essential for disrupting superbugs (e.g., MRSA), bacterial biofilms, and viruses such as human immunodeficiency virus 1 (HIV-1) and respiratory syncytial virus (RSV). In the central helix, the central arginine R23 is of particular importance in binding to bacterial membranes or DNA. Mapping the functional roles of the cationic amino acids of the major antimicrobial region of LL-37 provides a basis for designing antimicrobial peptides with desired properties. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Keywords: Antimicrobial peptide; Cathelicidin; LL-37; Membranes; NMR; Structure–activity relationship.

Fig. 1

The processing pathways (A) and multiple functions (B) of human cathelicidin peptides. In human sweat, LL-37 can be processed into shorter peptides such as KR-20, LL-23, and KS-30 [35]. It is unclear whether ALL-38 can be processed in a similar manner in the human reproductive system [34].

Fig. 2

A cartoon view of the membranes of (A) Gram-negative bacteria and (B) Gram-positive bacteria. The membranes of Gram-negative bacteria are composed of two layers: the outer membrane rich in LPS and the inner membrane rich in anionic PG. Gram-positive bacteria have a cell wall consisting of lipoteichoic acid and peptidoglycan and a cytoplasmic membrane.

Fig. 3

Ribbon diagrams of the three-dimensional structures of human LL-37 and its fragments bound to membrane-mimetic micelles. (A) An ensemble of 5 structures of LL-37 is shown. The structured region of LL-37 superimposes well but not the disordered C-terminal tail. Also presented are NMR structures of LL-12 (B), IG-25 (C), GI-20 (D), GF-17 (E), and KR-12 (F). For clarity, only the side chains of GF-17 and KR-12 are shown and labeled. The structures of LL-37 (PDB entry: 2K6O), LL-12 (PDB entry: 2FBU), IG-25 (PDB entry: 2FCG), and GF-17 (PDB entry: 2L5M) were determined in the presence of SDS micelles [21, 56, 61], while the structures of GI-20 and KR-12 were determined in complex with D8PG [56, 57]. In several cases including LL-37 itself, we found a similar structure for the peptide in SDS and D8PG [56]. Note the conformational changes at the C-terminus of LL-12 (B) and at the N-terminus of IG-25 (C) compared to the conformation of intact LL-37 (A).

Fig. 4

The central helix stands out in the high-quality 3D structure of human LL-37 as the key antimicrobial region. (A) While the C-terminal tail does not participate in membrane binding, S9 splits the membrane-targeting hydrophobic surface (gold) of the long amphipathic helix (residues 2-31) into two domains: an N-terminal short helix and a long central helix. Such a structure explains the cooperative binding of LL-37 to LPS [69]. It also indicates that the central helix is the key antimicrobial region of LL-37, which is responsible for antibacterial, anti-biofilm, and antiviral activities discussed in this article. (B) Identification of important side chains of LL-37 directly involved in membrane interactions by NMR. All of the four hydrophobic aromatic rings of F5, F6, F17, and F27 (green), as well as cationic R23 (stick), interact with phosphatidylglycerols. In this view, only the side chain of R23 is close to the hydrophobic surface (gold). It is thus an important interfacial basic side chain required for membrane interactions or binding to other anionic components such as DNA.