PA1b, an Insecticidal Protein Extracted from Pea Seeds (Pisum sativum):  ¹H-2-D NMR Study and Molecular Modeling^,

Abstract

PA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world. As such, PA1b is the first genuine protein phytotoxin specifically toxic to insects, which makes it a promising tool for seed weevil damage control. We have determined the 3-D solution structure of PA1b, using 2-D homonuclear proton NMR methods and molecular modeling. The primary sequence of the protein does not share similarities with other known toxins. It includes six cysteines forming three disulfide bridges. However, because of PA1b resistance to protease cleavage, conventional methods failed to establish the connectivity pattern. Our first attempts to assign the disulfide network from NOE data alone remained unsuccessful due to the tight packing of the cysteine residues within the core of the molecule. Yet, the use of ambiguous disulfide restraints within ARIA allowed us to establish that PA1b belongs to the inhibitor cystine-knot family. It exhibits the structural features that are characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first to the second strand and a series of turns. A comparison of the structural properties of PA1b with that of structurally related proteins adopting a knottin fold and exhibiting a diverse range of biological activities shows that the electrostatic and lipophilic potentials at the surface of PA1b are very close to those found for the spider toxin ACTX-Hi:OB4219, thereby suggesting activity on ion channels.

Molecular Requirements for the Insecticidal Activity of the Plant Peptide Pea Albumin 1 Subunit b (PA1b)*

“…Except for these homologues, the sequence of this protein does not share any similarity with those of other known toxins. The three-dimensional structure, resolved from the extracted peptide (9), shows that PA1b adopts a typical knottin fold with a triple-stranded antiparallel ␤-sheet and three buried interlocked disulfide bonds, a signature of the inhibitory cystine knot family (10,11). The ring embedded in the structure, through which the third disulfide bond threads, is composed of eight residues.…”

New Mode of Action for a Knottin Protein Bioinsecticide

“…The three-dimensional structure resolved from the extracted peptide shows that PA1b adopts a typical knottin fold with a triple-stranded antiparallel ␤-sheet and three buried interlocked disulfide bonds, the signature of the inhibitory cystine knot (ICK) 2 family (8). The ICK motif is present in a large number of fungal, plant, and animal peptides and provides them with proteolytic, thermal, and chemical stability.…”

“…Although responsible for a wide range of biological activities (9), ICK peptides are mainly voltage-gated ion channel blockers when extracted from animals and enzyme inhibitors when they are of plant origin (10). Structural analyses have also revealed similarities between PA1b and the ICK atracotoxin ACTX-Hi: OB4219, which is isolated from the insectivorous Australian funnel-web spider Hadronyche infensa and which targets an unknown channel (8,11).…”

Legume cyclotides shed light on the genetic origin of knotted circular proteins

“…Poth et al (11) present evidence supporting a gradual evolutionary path, whereby the albumin-1 chainb slowly evolved into a more stable cyclotide domain. For example, the pea albumin-1 subunit-b (PA1b), one of the best-studied Fabaceae albumin components, is a 37-aa peptide from pea seeds (Pisum sativum), which also contains a cystine-knot structure (13). Remarkably, the cystine-knot core of PA1b overlays extremely well with that of the cyclotide Cter M. The composition and size of the PA1b loops are, however, totally different from those of the cyclotide Cter M. Recent mutagenesis studies on PA1b have also recently shown that this albumin domain is highly tolerant to mutations outside the cystine knot core (14).…”

“…As indicated by Poth et al (11), the structural analysis of PA1b may reveal some clues about how this could have happened. The NMR structure of PA1b (13) reveals that the N and C termini are very close to each other, and it is possible that mutations in the albumin genes predisposed them to cyclization during the evolution process.…”