Solution Structure of PAFP-S:  A New Knottin-Type Antifungal Peptide from the Seeds of Phytolacca americana^,

Abstract

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1 H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 Å for the backbone heavy atoms and 2.19 Å for all heavy atoms, respectively. For the well-defined triple-stranded -sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 Å. The global fold involves a cystine-knotted three-stranded antiparallel -sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four -reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.

The hydrophobic surface of PaAMP from pokeweed seeds is essential to its interaction with fungal membrane lipids and the antifungal activity

“…DmAMP is predicted to have a same structure of RaAFP2 [2] that is comprised of a babb fold stabilized by 4 disulfide bonds while PaAMP contains a cysteine-knot fold [5,12]. Further study is needed to know the exact nature of the fungal sphingolipids that specifically interact with PaAMP.…”

“…The structure of the PaAMP protein determined by NMR [12] has cysteine-knot folding with anti-parallel b-sheets stabilized by three disulfide bridges and it has a positive patch and a hydrophobic surface [5,12]. The two structural domains are so arranged to each other that the protein is strongly amphipathic.…”

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions

“…This knotted structure, also referred to as cystine knot, provides an extraordinary stability to peptides and has also been described for CYCLOTIDES (see below). KNOTTIN-type peptides, found in Mirabilis jalapa (Mj-AMP1 and Mj-AMP2) and Phytolacca americana (PAFP-S), are derived from a preprotein and display mainly antifungal activity Gao et al, 2001;de Souza Cândido et al, 2014). Interestingly, PA1b, a KNOTTIN from pea, is embedded in albumin PA1.…”

New Antimicrobial Agents of Plant Origin

“…The threedimensional structure of the P. americana peptide was determined by NMR (Gao et al 2001). It has an antiparallel b-sheet formed by three strands (Fig.…”