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

Abstract

PA1b (pea albumin 1, subunit b) is a small and compact 37-amino acid protein, isolated from pea seeds (Pisum sativum), that adopts a cystine knot fold. It acts as a potent insecticidal agent against major pests in stored crops and vegetables, making it a promising bioinsecticide. Here, we investigate the influence of individual residues on the structure and bioactivity of PA1b. A collection of 13 PA1b mutants was successfully chemically synthesized in which the residues involved in the definition of PA1b amphiphilic and electrostatic characteristics were individually replaced with an alanine. The three-dimensional structure of PA1b was outstandingly tolerant of modifications. Remarkably, receptor binding and insecticidal activities were both dependent on common well defined clusters of residues located on one single face of the toxin, with Phe-10, Arg-21, Ile-23, and Leu-27 being key residues of the binding interaction. The inactivity of the mutants is clearly due to a change in the nature of the side chain rather than to a side effect, such as misfolding or degradation of the peptide, in the insect digestive tract. We have shown that a hydrophobic patch is the putative site of the interaction of PA1b with its binding site. Overall, the mutagenesis data provide major insights into the functional elements responsible for PA1b entomotoxic properties and give some clues toward a better understanding of the PA1b mode of action.

Discovery of an unusual biosynthetic origin for circular proteins in legumes

“…Like kB1 (33), PA1b recently has been demonstrated to be highly tolerant to modification outside the cystine knot core (32). This observation supports the possibility of divergent evolution of cyclotides from ancestral albumin domains.…”

“…SPR studies done here show that Cter M interacts with membrane lipids, a mechanism common to other cyclotides, which appear to not have stereospecific receptors and are instead pore-forming membrane disruptors (34). In contrast, PA1b interacts specifically with a membrane-based receptor in insect cells (32).…”

New Mode of Action for a Knottin Protein Bioinsecticide

“…The absence of any effect of PA1b when directly applied to the inside of the cell corroborates previous results showing that PA1b is internalized in Sf9 cells but that it has an effect only when the toxin is localized outside of the cell (17). However, the most important observation is the absence of any effect of the F10A mutant, which was demonstrated as being without toxicity and without any indication of binding to the PA1b receptor (20). Hence, it is clear that the reported effect of PA1b in blocking an ionic current is very specific to the PA1b structure, and this could underlie its toxicity.…”

“…The F10A mutant was obtained by chemical synthesis according to the method described previously (20).…”

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

“…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). These observations support the possibility of divergent evolution of cyclotides from ancestral albumin domains, wherein evolution and natural selection provided an alternative loop decoration of the original cystine knot albumin core.…”