Unnatural Amino Acids with Abbreviations
1.2. Classification of AMPs
1.2.2. Classification According to Primary Structure
1.2.2.1. An Unusual Proportion of Proteinogenic Amino Acids:
A high proportion of certain amino acids in a specific primary structure leads to the folding of the sequences, which is different from regular helices and sheets structure. These types of AMPs are discussed below:
1.2.2.1.1. Defensins:
Defensins are cationic AMP rich in Arg and Cys residues with a -hairpin structure stabilized by three to four disulfide bonds (Figure 1.6). It binds to the membrane to form pore-like membrane defects that lead to efflux of the intracellular matrix, causing cell death. Plant defensins have antimicrobial, α-amylase,154 and trypsin inhibitory activities154 with activity against yeast, oomycetes, and necrotophic pathogens.155,156 Human defensins are of three types- α, β, θ. There are six subclasses of α defensins containing three conserved disulfide connectivities between Cys I−VI, Cys II−IV, and Cys III−V. 157 β-defensins are longer in sequence than the α defensins and have Cys I−V, Cys II−IV, Cys III−VI disulfide bond connectivities. There are different subclasses of human β-defensins (hBD). The hBD1 and hBD2 are rendered inactive in the presence of physiological salt concentrations.158,159 The hBD3 retains its activity against Staphylococcus aureus (S. aureus) and vancomycin-resistant Enterococcus faecium at physiological salt concentrations.160 Insect defensins are about 29–34 amino acids long, active against gram-positive and gram-negative bacteria.161 However, the virulence against the gram- positive bacteria (S. aureus) is usually more than the gram-negative family.162
1.2.2.1.2. Protegrins:
Protegrins are a class of AMPs similar to defensins and contain 16 to 18 amino acid residues.
Protegrins are highly active against both Gram-positive and Gram-negative bacteria, chlamydia, mycobacteria and viruses like HIV-1.169,170
Figure 1.6. Secondary structures of (a) Human α-defensin 1 derivative (2PM5)163, (b) BNBD- 12 bovine neutrophil -defensin (1BNB)164, (c) Insect defensin (1ICA)165, (d) β-defensin HBD-1(1IJV)166, (e) Raphanus sativus Antifungal Protein 1 (Rs-AFP1) plant defensin (1AYJ)167 and (f) theta-defensin HTD-2 (2LZI)168
1.2.2.1.3. Histatins:
Histatins contain a group of neutral and basic His-rich peptides (Figure 1.7) present in secretions of human submandibular and parotid glands and human saliva.55 The former contains Histatin 5, and the later contains histatin 1 and 3.171 All three histatins can kill pathogenic yeast C. albicans. They are unstructured in aqueous solutions, but they form α helical conformation in the polar protic solvent system. Due to low amphipathicity, histatins are non-cytotoxic compared with other AMPs. Calcitermin C is a 15 residue AMP containing
three His residues at the N-terminal that adopts a helical conformation in the membranes.172 It is found in human airways and is effective against gram-negative bacteria.
Figure 1.7. Primary sequence for histatin-1, histatin-2, histatin-3, and histatin-5, which are most frequently studied for their antimicrobial activity.
1.2.2.1.4. Cathelicidins:
Cathelicidins are short cationic peptides that belong to the family HDP found in the innate immune system. The mature protein was isolated from the neutrophils, human skin and sweat.
Cathelicidins have a 100 amino acid long conserved N-terminal pre-pro region called the cathelin domain (Figure 1.8a).173 Initially, the cathelin domain was studied for its antimicrobial activity, but nowadays, it is more appreciated for its immunomodulatory functions. The mature cathelicidin peptides vary with both amino acid size and sequences, leading to considerable differences in their 3D structure. Cathelicidins contain α-helices, β-hairpins, extended networks or form cyclic peptides (Figure 1.8b). It is found in nearly all vertebrates. While in some species, like mouse (CRAMP), dog (K9CATH), and humans (LL-37), only one cathelicidin has been identified, multiple cathelicidins are found in chicken, horse, pig, and cattle. The primary source of LL-37 in humans are neutrophils, which store the inactive pro-peptide in their secretory granules and secrete upon activation. However, other cell types, including
macrophages, epithelial cells, lymphocytes, and keratinocytes, also produce Cathelicidins.174-
180
Figure 1.8. (a) Schematic representation of the gene and propeptide of the Cathelicidin in vertebrate.181-186 Color codes indicate the exons and their corresponding translated peptide fragments. (b) The secondary structure of Cathelicidins include α-helical, β-sheet, extended helical, and loop structure, which were predicted online by the Ressource Parisienne en BioInformatique Structurale web portal.187 This figure has been adopted from Ref. 173 (open access) with modification.
1.2.2.2. AMPs Containing Uncommon Modified Amino Acid:
These types of peptides are isolated from bacteria and fungi, which are more similar to conventional antibiotics than the AMPs formed in the higher organisms. Lantibiotics and peptaibols are the two classes of peptides considered here.
1.2.2.2.1. Lantibiotics:
They are polypeptide antibiotics containing uncommon modified amino acids with a small ring structure enclosed with a thioether bond. Lantibiotics are ribosomally synthesized and post- translationally modified peptides. These AMPs are well studied due to their commercial uses in the food industries for making dairy products. Lantibiotics are produced by many gram- positive bacteria like Streptococcus and Streptomyces to attack other gram-positive bacteria.
The duramycin family of lantibiotics bind phosphoethanolamine in the membrane of its target cells and disrupts different physiological functions. One peptide of this family is nisin, which binds to lipid II, a cell wall precursor lipid component of target bacteria and disrupts cell wall formation (Figure 1.9a).188,189 NMR studies reveal that it is flexible in water but forms a β-turn structure upon binding to the micelles. Activity of these AMPs are not related to any regular secondary structures like helices and sheets.189
Figure 1.9. (a) Primary structure of nisin. The unusual amino acids present in the primary structure are dehydroalanine (Dha), dehydrobutyrine (Dhb) and L-2-aminobutyric acid (Abu).
(b) Primary structure of peptaibol Alamenthicin isolated from Trichoderma viride which contains high percentages of unnatural amino acid residue amino isobutyric acid (Aib).
1.2.2.2.2. Peptaibols:
This is an interesting class of AMPs which contain a large proportion of unnatural amino acid residue Amino isobutyric acid (Aib). Aib is a conformationally restricted amino acid residue that forms α helical structure. Alamenthicin is a well-known peptaibol containing 20 amino acid residues, isolated from the fungus Trichoderma viride (Figure 1.9b). It forms a α-helical conformation in non-aqueous solvent system as well as in lipid membrane bilayers.190