Binding of the AVR4 Elicitor of Cladosporium fulvum to Chitotriose Units Is Facilitated by Positive Allosteric Protein-Protein Interactions: THE CHITIN-BINDING SITE OF AVR4 REPRESENTS A NOVEL BINDING SITE ON THE FOLDING SCAFFOLD SHARED BETWEEN THE INVERTEBRATE AND THE PLANT CHITIN-BINDING DOMAIN

Harrold A Van Den Burg,Christian A.E.M Spronk,Sjef Boeren,Matthew A Kennedy,Johannes P.C Vissers,Geerten W Vuister,Pierre J.G.M De Wit,Jacques Vervoort

doi:10.1074/jbc.m312594200

Harrold A Van Den Burg, Christian A.E.M Spronk + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m312594200

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Abstract

The attack of fungal cell walls by plant chitinases is an important plant defense response to fungal infection. Anti-fungal activity of plant chitinases is largely restricted to chitinases that contain a noncatalytic, plant-specific chitin-binding domain (ChBD) (also called Hevein domain). Current data confirm that the race-specific elicitor AVR4 of the tomato pathogen Cladosporium fulvum can protect fungi against plant chitinases, which is based on the presence of a novel type of ChBD in AVR4 that was first identified in invertebrates. Although these two classes of ChBDs (Hevein and invertebrate) are sequentially unrelated, they share structural homology. Here, we show that the chitin-binding sites of these two classes of ChBDs have different topologies and characteristics. The K(D), DeltaH, and DeltaS values obtained for the interaction between AVR4 and chito-oligomers are comparable with those obtained for Hevein. However, the binding site of AVR4 is larger than that of Hevein, i.e. AVR4 interacts strictly with chitotriose, whereas Hevein can also interact with the monomer N-acetylglucosamine. Moreover, binding of additional AVR4 molecules to chitin occurs through positive cooperative protein-protein interactions. By this mechanism AVR4 is likely to effectively shield chitin on the fungal cell wall, preventing the cell wall from being degraded by plant chitinases.

Highlights

The attack of fungal cell walls by plant chitinases is an important plant defense response to fungal infection
Current data confirm that the race-specific elicitor AVR4 of the tomato pathogen Cladosporium fulvum can protect fungi against plant chitinases, which is based on the presence of a novel type of chitin-binding domain (ChBD) in AVR4 that was first identified in invertebrates
Six Cys residues are conserved in the CBM14 motif, and we showed recently that the corresponding Cys residues in AVR4 are involved in a disulfide bond pattern similar to that found in Tachycitin [10]

Summary

EXPERIMENTAL PROCEDURES

Materials—AVR4 was produced in culture by the methylotrophic yeast Pichia pastoris and purified from culture fluids [37]. The fluorescence quenching at full saturation of binding (Fρ) was estimated by plotting 1/(F0 Ϫ F) versus 1/[S], and extrapolating to the y axis, where F0 is the fluorescence intensity of AVR4 without ligand, and F is the fluorescence intensity of AVR4 at the chito-oligomer concentration [S]. Calibration of the TOF analyzer was performed with a CsI solution of 2 mg/ml in isopropanol/water (50:50, v/v) over the mass range of 800 – 7100 Da. Nuclear Magnetic Resonance Spectroscopy—The NMR samples contained typically 1.5 mM 13C/15N AVR4 dissolved in 20 mM acetate-d4, pH. Triple and double resonance heteronuclear NMR experiments performed to obtain backbone and side chain assignments of AVR4 included three-dimensional HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, (H)CCH-TOCSY, and HC(C)H-TOCSY (Protein Pack; Varian Inc.). NMR Titration Experiments—Binding of chito-oligomers to AVR4 was followed by recording 1H-15N HSQC spectra at five different temperatures. Thermodynamic parameters (⌬H and ⌬S) were estimated from a van’t Hoff plot based on a set of KA values obtained from a set of backbone resonances

RESULTS AND DISCUSSION

Scatchardc KD

Maximum quenching of F

Stoichiometrya kDa