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.

Background: c-Abl regulates cell signaling and participates in leukemia pathogenesis via Bcr-Abl chimeric protein.Results: N-Cap and SH3 residues acquire μs-ms motions within the regulatory unit and membrane anchoring upon protein activation.Conclusion: N-Cap-myristoyl tether triggers c-Abl to anchor membrane because of μs-ms dynamics within this regulatory region.Significance: Binding to the membrane is lost in Bcr-Abl chimeric protein, which underlies leukemia.

In spite of the effectiveness of Imatinib for chronic myeloid leukemia (CML) treatment, resistance has repeatedly been reported and is associated with point mutations in the BCR-ABL chimeric gene. To overcome this resistance, several inhibitors of BCR-ABL tyrosine kinase activity were developed. In this context, computational simulations have become a powerful tool for understanding drug-protein interactions. Herein, we report a comparative molecular dynamics analysis of the interaction between two tyrosine kinase inhibitors (imatinib or nilotinib) against wild type c-ABL protein and 12 mutants, using the semi-empirical linear interaction energy (LIE) method, to assess the feasibility of this approach for studying resistance against the inhibitory activity of these drugs. In addition, to understand the structural changes that are associated with resistance, we describe the behavior of water molecules that interact simultaneously with specific residues (Glu286, Lys271 and Asp381) of c-ABL (wild type or mutant) and their relationship with drug resistance. Experimental IC50 values for the interaction between imatinib, wild type c-ABL, and 12 mutants were used to obtain the proper LIE coefficients (α, β and γ) to estimate the free energy of the binding of imatinib with wild-type and mutant proteins, and values were extrapolated for the analysis of the nilotinib/c-ABL interaction. Our results indicate that LIE was suitable to predict the superior inhibitory activity of nilotinib and the resistance to inhibition that was observed in c-ABL mutants. Additionally, for c-ABL mutants, the observed number of water molecules being turned over while interacting with amino acids Glu286, Lys271 and Asp381 was associated with resistance to imatinib, resulting in a less effective inhibition of the kinase activity.

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