Notice: Wiley Online Library will be unavailable on Saturday 27th February from 09:00-14:00 GMT / 04:00-09:00 EST / 17:00-22:00 SGT for essential maintenance. Apologies for the inconvenience.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
In response to fungal invasion and other signals, plants accumulate a number of proteins that are involved in defense against pathogens.1 Among these proteins, pathogenesis-related (PR) proteins are grouped into families based on primary structure, serological relatedness, and enzymatic and biological activities.2 Osmotin is a 24 kDa protein belonging to the PR-5 protein family whose members are homologous to the sweet-tasting protein thaumatin. Osmotin and other PR-5 proteins were shown to have antifungal activity in vitro against a broad range of fungi, including several plant pathogens.1
PR-5 proteins can be categorized into three subclasses based on their isoelectric point (pI): acidic, neutral and basic.3 Comparative analysis of the primary structure of osmotin, which is a basic PR-5 protein from tobacco, and several other PR-5 proteins reveal several interesting features.4 The alanine located at the cleavage site of N-terminal leader sequence and 16 cysteine residues, which are distributed throughout the protein and linked via formed disulfide-bridges, are conserved spatially among virtually all PR-proteins.4 The fungal growth inhibition by osmotin and zeamtin, a maize PR-5 protein, is correlated with plasma membrane permeabilization and dissipation of the plasma membrane potential,5, 6 suggesting a physical interaction between PR-5 proteins and the plasma membrane of sensitive fungi. In contrast, it is proposed that specific target receptors on the membrane determine the sensitivity or resistance to PR-5 proteins.7 However, the precise mechanism by which osmotin interacts with specific fungal pathogens and mediates plasma membrane permeability has not been clearly elucidated. Recent genetic and biochemical data indicate that osmotin utilizes a signal transduction pathway in yeast to increase the susceptibility of a target fungus to its cytotoxic effects.8 To understand the structural basis of antifungal activity of osmotin, we have determined the crystal structure of tobacco osmotin at 2.3 Å resolution and compared its structure with other antifungal proteins and thaumatin.
Materials and Methods.
Osmotin was purified from salt-adapted tobacco cell suspension cultures (Nicotiana tabacum L. var. Wisconsin 38) to apparent homogeneity as described.9 Lyophilized osmotin was dissolved in 25 mM N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES; pH 7.5) to the concentration of 7 mg/mL. Crystals were obtained by the vapor diffusion method at 22°C in hanging drops containing a 1:1 mixture of a protein solution with reservoir solution (0.1 M Tris-HCl, pH 8.5, 0.4 M lithium sulfate, 0.06 M nickel chloride). Osmotin crystals belong to space group P1 with unit cell dimensions of a = 41.78 Å, b = 41.79 Å, c = 59.56 Å, α = 100.84°, β = 92.41°, γ = 96.57°. There are two monomers in an asymmetric unit, with the corresponding Vm of 2.27 Å3/Da and the solvent content of 45%. The crystal diffracted to 2.3 Å resolution using synchrotron X-rays at Brookhaven National Laboratory. A total of 26,588 reflections were measured, which were merged to 14,549 unique reflections with Rsym of 7.4%. The merged data set is 83.4% complete to 2.3 Å. The diffraction images were processed with the HKL package.
The structure of osmotin was solved by molecular replacement using the EPMR program with tobacco PR-5d [Protein Data Bank (PDB) ID code: 1AUN)10 as the probe and refined using the CNS with noncrystallographic symmetry (NCS) restraints. The refined model consists of 3102 nonhydrogen atoms from 410 amino acid residues and 140 water molecules in an asymmetric unit. The crystallographic R/ Rfree values are 20.8%/25.8% for all reflections in the range of 30–2.3 Å. The average B-factor is 19.9 and 20.5 Å2 for main-chain and side-chain atoms, respectively. The model has good geometry, with no residues in the disallowed regions of the Ramachandran plot. The coordinate of osmotin is deposited in the PDB with the accession code 1PCV. Data processing and refinement statistics are summarized in Table I.
Table I. Data Collection and Refinement Statistics
Brookhaven National Laboratory.
Rsysm = ΣhΣ i| I(h)i < I(h) >|/ΣhΣ iI(h)i, where I(h) is the intensity of reflection h, Σ h is the sum over all reflections, and Σ i is the sum over the i measurements of reflection h.
The osmotin crystals have a noncrystallographic dimer in the asymmetric unit. The two monomers have slightly different tertiary structures. The root- mean-square deviation (RMSD) of backbone atoms between the two monomers is 0.016 Å. Osmotin is composed of three domains and shows a similar fold with thaumatin11 and other PR-5 proteins such as zeamation12 and tobacco PR-5d protein10 (Fig. 1). Domain I consists of an 11-strand, flattened β-sandwich (residues 1–53, 82–125, and 175–205) that forms the compact core of molecule. Domain II consists of several loops extending from domain I and is stabilized by four disulfide bonds (residues 126– 174). Domain III consists of a small loop (residues 54–81) with two disulfide bonds. Osmotin has a pronounced cleft formed by domains I and II (Fig. 1).
In general, the tertiary structure of osmotin is homologous to those of thaumatin and other antifungal PR-5 proteins (zeamatin and tobacco PR-5d). Most of the differences among them are in domain II, and the most notable of these is the absence of thaumatin loop in antifugal PR-5 proteins [Fig. 1(B)]. The thaumatin loop (Asp-Ala-Ala-Leu-Asp-Ala- Gly) has a unique sequence similar to peptide-sweetners (L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester), which are postulated as one of the important sweet-taste determinants13 and located in the same side of the cleft region. They also show the difference in surface charge distribution (Fig. 2). Analysis of the electrostatic properties of the osmotin monomer reveals that the cleft region formed by domains I and II is highly acidic (Fig. 2). The acidic residues involved in the formation of the acidic cleft of osmotin are Glu84, Asp97, Asp102, and Asp185, which extend into the cavity of the cleft [Fig. 1(A)]. As reported previously, tobacco PR-5d and zeamatin also have an acidic cleft,10, 12 whereas thaumatin has a basic surface in the cleft region.11 However, backsides of the clefts of osmotin, tobacco PR-5d, zeamatin, and thaumatin are predominantly basic in common. Therefore, the charge difference between antifungal PR-5d proteins and sweet-tasting thaumatin is localized only in the cleft. Phosphopyridoxylation of lysine residues of thaumatin (78, 97, 106, 137, 187) around the cleft, which corresponds to the acidic cleft in osmotin-like proteins, reduced sweetness and dephosphorylation of modified lysine residues restored sweetness except for lysine 106 located at the center of the cleft.14 It indicates that the positive charges on these lysine residues of the cleft region may play an important role in sweetness by a multipoint interaction with a putative thaumatin receptor. Considering the sequence and structural homology among thaumatin and PR-5 proteins, interaction between thaumatin and its receptor might also be conserved in PR-5 proteins and their putative receptors. The presence of osmotin receptor on the cell surface is also suggested.7, 8 Therefore, it can be assumed that the acidic cleft of osmotin may also be involved in interaction with its receptor in the plasma membrane of fungi. However, the distinct differences of surface properties between antifugal PR-5 proteins and sweet-tasting thaumatin are related to their differences in molecular functions.
Although all of the known antifungal PR-5 proteins are similar in their overall structures and in the acidic cleft regions [Figs. 1(B) and 2], they show different activities and specificities against their target cells.7, 15 Based on the assumption that the acidic cleft is important for its antifungal activity and is relevant to receptor binding, differences of topology and surface electrostatic potential around the cleft are considered to determine the specificity of PR-5 proteins to their target cells. Chemical modification of residues around the acidic cleft is needed to elucidate the precise mode of antifugal activity of osmotin.
We thank Dr. Robert Sweet and scientists at Beamline 12C of the National Synchrotron Light Source, USA, for assistance during data collection.