Solution Structure of Synthetic Penaeidin-4 with Structural and Functional Comparisons with Penaeidin-3

Abstract

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.Antimicrobial peptides (AMPs) 1 can form a physiochemical barrier that deters infection by potential pathogens in an individual organism. This is true for many multicellular organisms including both vertebrate and invertebrate animals (1-4). Many families of AMPs have been identified, and some are of particular interest for direct application to human health (5-7). In addition to direct therapeutic application of native AMPs, the potential exists for the enhancement of activity through the use of synthetic peptide derivatives that incorporate atypical or nonnative amino acids, so as to resist natural metabolic proteases, enhance pathogen specificity, and increase effectiveness (8 -10).Penaeidins are known to be a diverse family of cationic peptides that, despite their extensive variability in primary structure, target in an almost exclusive manner Gram(ϩ) bacteria and fungi (11, 12). Variability in primary structure has been noted in the form of multiple classes (designated 1/2, 3, and 4) and multiple isoforms of some classes (13).The typical penaeidin molecule is composed of two domains, an unconstrained proline-rich N-terminal domain (PRD) and a cysteine-rich domain (CRD) with a stable ␣-helical structure...

Adaptive evolution after duplication of penaeidin antimicrobial peptides

“…Although ML-based codon substitution analyses showed the evidence of positive Darwinian selection in both domains of penaeidin, a relatively more number of positively selected codon sites were observed in PRD than the CRD. It could be possible that CRD is relatively more conserved than the PRD [10,12]; therefore, more amino acid residues in PRD are subjected to natural selection in different environmental Table 2), respectively. The structure was displayed using RasMol V2.7.2.1.1 (http://www.…”

“…Although both domains are under the influence of positive Darwinian selection, the differences in the number of positively selected amino acid residues in both domains could be attributed to the structural organization of the respective domains. For example, the N-terminal proline rich residues of penaeidin are less conserved, whereas the cysteine rich C-terminal domain is stabilized by three conserved disulphide bonds [10,12,24], therefore, more number of positively selected amino acid residues in PRD is expected. The high sequence variability that have generated by gene duplication events followed by the positive Darwinian selection among the PRD of different classes of penaeidin may have resulted in variation in target specificity and gain in antimicrobial function [10,12].…”

“…For example, the N-terminal proline rich residues of penaeidin are less conserved, whereas the cysteine rich C-terminal domain is stabilized by three conserved disulphide bonds [10,12,24], therefore, more number of positively selected amino acid residues in PRD is expected. The high sequence variability that have generated by gene duplication events followed by the positive Darwinian selection among the PRD of different classes of penaeidin may have resulted in variation in target specificity and gain in antimicrobial function [10,12]. On contrary, the CRD, which is relatively conserved, might have a tandem or synergistic role [10,12].…”

“…The high sequence variability that have generated by gene duplication events followed by the positive Darwinian selection among the PRD of different classes of penaeidin may have resulted in variation in target specificity and gain in antimicrobial function [10,12]. On contrary, the CRD, which is relatively conserved, might have a tandem or synergistic role [10,12].…”

Diversity in penaeidin antimicrobial peptide form and function

“…Currently two models have been put forth for anti-MP mechanism of action [2,5,33] which are the barrel-like (barrel-stave) pore forming mechanism and the carpeting mechanism. Each of these would result in the compromising of the microbial membrane, and much evidence corroborating both of these mechanisms has been reviewed previously [2,5,33].Penaeidins are antimicrobial peptides from shrimp that are unique in that they are composed of two very different domains [34][35][36], an N-terminal proline-rich domain (PRD) linked to the C-terminal cysteine-rich domain (CRD) [37,38]. While the PRD forms an extended structure, the folded structure of CRD contains an α-helix stabilized by disulfide bonds [37,38].…”