Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin‐1 and fowlicidin‐2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin‐3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin‐3 comprises 27 amino‐acid residues and adopts a predominantly α‐helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram‐negative and Gram‐positive bacteria in vitro, including antibiotic‐resistant strains, with minimum inhibitory concentrations in the range 1–2 µm. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin‐3 retains bacteria‐killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide‐induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 µm. Fowlicidin‐3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic‐resistant pathogens.

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti‐infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad‐spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858–2867). Here we report that fowlicidin‐1 mainly adopts an α‐helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin‐1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)‐binding activities. The short C‐terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16–23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5–7) in the N‐terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin‐1 analog, with deletion of the second N‐terminal segment (residues 5–7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti‐infective agent.

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.