1. PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum): 1H-2-D NMR study and molecular modeling
Laurence Jouvensal, Laurence Quillien, Eric Ferrasson, Yvan Rahbé, Jacques Guéguen, Françoise Vovelle Biochemistry. 2003 Oct 21;42(41):11915-23. doi: 10.1021/bi034803l.
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.
2. Solution structure of Eucommia antifungal peptide: a novel structural model distinct with a five-disulfide motif
Ren-Huai Huang, Ye Xiang, Guan-Zhong Tu, Ying Zhang, Da-Cheng Wang Biochemistry. 2004 May 25;43(20):6005-12. doi: 10.1021/bi036263y.
The three-dimensional structure in aqueous solution of Eucommia antifungal peptide 2 (EAFP2) from Eucommia ulmoides Oliv was determined using (1)H NMR spectroscopy. EAFP2 is a newly discovered 41-residue peptide distinct with a five-disulfide cross-linked motif. This peptide exhibits chitin-binding activity and inhibitory effects on the growth of cell wall chitin-containing fungi and chitin-free fungi. The structure was calculated by using torsion angle dynamic simulated annealing with a total of 614 distance restraints and 16 dihedral restraints derived from NOESY and DQF-COSY spectra, respectively. The five disulfide bonds were assigned from preliminary structures using a statistical analysis of intercystinyl distances. The solution structure of EAFP2 is presented as an ensemble of 20 conformers with a backbone RMS deviation of 0.65 (+/-0.13) A for the well-defined Cys3-Cys39 segment. The tertiary structure of EAFP2 represents the first five-disulfide cross-linked structural model of the plant antifungal peptide. EAFP2 adopts a compact global fold composed of a 3(10) helix (Cys3-Arg6), an alpha-helix (Gly26-Cys30), and a three-strand antiparallel beta-sheet (Cys16-Ser18, Tyr22-Gly24, and Arg36-Cys37). The tertiary structure of EAFP2 shows a chitin-binding domain (residues 11-30) with a hydrophobic face and a characteristic sector formed by the N-terminal 10 residues and the C-terminal segment cross-linked through the unique disulfide bond Cys7-Cys37, which brings all four positively charged residues (Arg6, Arg9, Arg36, and Arg40) onto a cationic face. On the basis of such a structural feature, the possible structural basis for the functional properties of EAFP2 is discussed.
3. Min-21 and min-23, the smallest peptides that fold like a cystine-stabilized beta-sheet motif: design, solution structure, and thermal stability
A Heitz, D Le-Nguyen, L Chiche Biochemistry. 1999 Aug 10;38(32):10615-25. doi: 10.1021/bi990821k.
Small disulfide-rich proteins provide examples of simple and stable scaffolds for design purposes. The cystine-stabilized beta-sheet (CSB) motif is one such elementary structural motif and is found in many protein families with no evolutionary relationships. In this paper, we present NMR structural studies and stability measurements of two short peptides of 21 and 23 residues that correspond to the isolated CSB motif taken from a 28-residue squash trypsin inhibitor. The two peptides contain two disulfide bridges instead of three for the parent protein, but were shown to fold in a native-like fashion, indicating that the CSB motif can be considered an autonomous folding unit. The 23-residue peptide was truncated at the N-terminus. It has a well-defined conformation close to that of the parent squash inhibitor, and although less stable than the native protein, it still exhibits a high T(m) of about 100 degrees C. We suggest that this peptide is a very good starting building block for engineering new bioactive molecules by grafting different active or recognition sites onto it. The 21-residue peptide was further shortened by removing two residues in the loop connecting the second and third cysteines. This peptide exhibited a less well-defined conformation and is less stable by about 1 kcal mol(-)(1), but it might be useful if a higher flexibility is desired. The lower stability of the 21-residue peptide is supposed to result from inadequate lengths of segments connecting the first three cysteines, thus providing new insights into the structural determinants of the CSB motif.
