Solution Structure of the Recombinant Penaeidin-3, a Shrimp Antimicrobial Peptide

Abstract

Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen3a analogue was determined by using two-dimensional 1 H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29 -58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys 32 -Cys 47 and Cys 48 -Cys 55 ). These two coils are in turn linked together by the third disulfide bond (Cys 36 -Cys 54 ). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg 45 and Arg 50 , which belong to the helix, and side chains of Arg 37 and Arg 53 , which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.Antimicrobial peptides are major elements of host-defense systems represented in all species from plants to vertebrate and invertebrate animals. Among these antimicrobial molecules, cysteine-rich peptides are the most widespread. They are structurally classified into (i) peptides with a ␤-sheet structure such as the mammalian defensins (1), (ii) peptides with a ␤-hairpin-like fold such as tachyplesins from horseshoe crabs (2), thanatin (3), porcine protegrins (4, 5), androctonin (6), or gomesin (7), and (iii) peptides adopting the cystine-stabilized ␣␤-motif, including invertebrate and plant defensins (8Ϫ10).Only recently, such effectors of innate immunity were isolated from crustaceans, whereas numerous peptides have been characterized from other arthropods, both insects and chelicerates. Three peptides, named penaeidins (Pen), 1 were initially purified in their active form (5.48Ϫ6.62 kDa) from the hemocytes of the shrimp Litopenaeus (Penaeus) vannamei, and they were fully characterized at the amino acid level (Pen-1, Pen-2, Pen3a) and by cDNA cloning from a hemocyte library (pen-2, -3a, -3b, and-3c) (11). Penaeidins are an original p...

Adaptive evolution after duplication of penaeidin antimicrobial peptides

“…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].…”

“…A total of 36 nucleotide sequences were retrieved from GenBank ( [6,7,11,13,16,18,22,24,25]; Table 1). Sequences were aligned using DAMBE version 4.5.2 [38,39] and BioEdit version 7.0.5.3 [40].…”

“…We used 3-dimensional (3D) structure of penaeidin-3 of Litopenaeus vannamei (Protein Data Bank number ¼ 1UEO; [24]; http://www.rcsb.org/pdb/) to map the positively selected sites in the 3D structure. The alignment of all classes of penaeidin with 1UEO amino acid residues is shown in Table 4.…”

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

“…Penaeidin-3 inhibited over 85% of the viral replication at 100 µM, with an EC 50 value of 1.56 ± 0.18, resulting in a selectivity index (SI = CC 50 /EC 50 ) of 64 (data not shown). Its unique mixed structure among the evaluated peptides (a linear proline-rich N-terminal fragment with a cyclic C-terminal fragment with three disulfide bonds) may have contributed to the detected activity, since another study has already described this feature (Yasin et al 2000, Yang et al 2003.…”

A folding-dependent mechanism of antimicrobial peptide resistance to degradation unveiled by solution structure of distinctin

“…The first three classes include molecules that are unstructured in water, or generically aggregated under drastic concentration͞ionic strength conditions, but present an ␣-helical conformation when interacting with hydrophobic media (1, 2). The fourth group includes examples presenting mainly or only ␤-sheet structure in aqueous solution (19,20). In this article, we report the 3D structure of D1, a peptide that here we propose as a prototype of a previously unrecognized class of antimicrobial derivatives.…”