Moniliophthora perniciosa Necrosis- and Ethylene-Inducing Protein 2 (MpNep2) as a Metastable Dimer in Solution: Structural and Functional Implications

Abstract

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.

A hypothesis to reconcile the physical and chemical unfolding of proteins

“…The SH2 domain from c-Abl protein and MpNep2 were purified and assigned as previously described (25,44). For the high-pressure fluorescence and NMR experiments, size exclusion chromatography in Superdex 75 10/300 (GE Life Science) was performed immediately before each experiment using 20 mM Tris·HCl (pH 7.4) containing 80 mM NaCl.…”

“…Experiments were performed three times with different preparations, and the results are expressed as the mean. Changes in the fluorescence spectra recorded at each pressure were quantified as the center of spectral mass (per centimeter) as previously described (44). Stability measurements were performed using urea, as previously reported for the MpNep2 protein (44).…”

“…S2). Because the changes in peak position were systematic and almost linear for pressure increases up to 2 kbar, sequential assignments were straightforward, made by following and extrapolating the earliest assignments (at room temperature and 1 bar) (44). We obtained an average 1 H chemical shift of 0.03 ± 0.06 ppm over all residues by comparing the values obtained at 2 kbar to those obtained at 1 bar for 15 and 25°C.…”

“…5A) at 25°C (unlike the results obtained by varying the pressure). Because NMR data indicate the presence of a unique cooperative decay upon treatment with urea, we designed singlepoint SAXS experiments under equilibrium using subdenaturing concentrations of urea, which were determined based on Trp fluorescence spectroscopy experiments (44), to probe conformational states that occur immediately before protein denaturation. The distance distribution function P(r) revealed an overall similar shape in the range of 0-0.75 M urea (Fig.…”

“…Increasing the urea concentration from 0.75 to 1 M led to an abrupt increase in the dimensional values, as observed for D max and R g , and a loss of protein structure; this finding reveals that urea, unlike pressure, appears to form a global cooperative ensemble of structures. We previously obtained a U 1/2 value of 1.41 M using Trp fluorescence spectroscopy (44). Using NMR spectroscopy, based on the average intensity of the cross-peaks in Fig.…”