Mechanism Of Bacterial Effector Protein Azurin Targeting Tumor Suppressor P53 And Inhibiting Its Ubiquitination

Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation.Dysfunction of p53 occurs frequently in human cancers.Thus,p53 becomes one of the most promising targets for anticancer treatment.Azurin,a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa.A bacterial effector protein Azurin triggers tumor suppression by stabilizing p53 and elevating its basal level.However,the structural and mechanistic basis of Azurin-mediated tumor suppression remains elusive.In this study,we focused on dissecting the structural basis of bacterial effector Azurin targeting on tumour suppressor p53 and inhibiting its ubiquitination.1.Structure of Azurin in complex with p53After exploration and optimization of protein expression conditions in E.coli,we have successfully reconstituted the homogeneous p53-DBD/ Azurin complexes with high expression and homogeneity in E.coli.After extensive screening and crystallization conditions,well-diffracted crystals were obtained.Eventually,the structure of p53-DBD in complex with Azurin was determined at 2.2 ? resolution.This p53-DBD/ Azurin complexes mode is consistent with the information we obtained from NMR titration.2.Interface of the Azurin-p53 complexHere we determined the atomic details of Azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance.The complex is stabilized by a distinct Azurin-p53 interface between one Azurin molecule and one p53-DBD molecule.The interface is mainly mediated by the p28 region.3.Molecular mechanism underpinning p53 stabilization by AzurinBased on our structural and pull-down data,several residues were identified as critical for the complex formation.Among them,most of the residues are located at Azurin p28 region.Notably,p28 peptide,an anti-glioma drug,specifically dampens the COP1-p53 binding and stabilizes p53 from degradation,and our structural analyses showed that there are overlaps between Azurin binding sites and reported ubiquitin E3 ligase COP1 binding sites on p53.Using in vitro protein ubiquitination assay,suggesting that Azurin might negatively regulate COP1-mediated p53 ubiquitination through competitively binding to p53.4.Structural-guided design of affinity-enhancing Azurin mutantThe transient and dynamic nature of p53-Azurin interaction has been the major hurdle to improve the anti-cancer efficacy and specificity of Azurin and its derived peptides.The structure of p53-Azurin complex reported here enables to guide structurebased mutant design to enhance the interaction.Accordingly,we designed a series of Azurin mutants that potentially stabilize the complex,and tested their alterations in p53 binding affinity using pull down assays and ubiquitination assay.These structureguided mutagenesis assays provided mutations of Azurin with significant affinity enhancement towards p53 and significantly decreased the ubiquitination level of p53.5.Affinity-enhancing mutations promote p53-regulated apoptosiswe examined the effect of Azurin as well as its mutant treatment on p53 mediated apoptosis.Apoptosis were increased significantly in cells treated with affinity-enhanced Azurin mutants.Our data demonstrated that cells treated with Azurin showed slightly increase in Bax and p21,and both of them were increased significantly in cells treated with affinity-enhanced Azurin mutants.Taking together,the present data provide atomic insight into the structural features underlying the mechanisms of p53 accumulation induced by a bacteria effector Azurin.Our structure-guided mutagenesis assays affirmed the structural and functional results,and provided new perspectives for designing Azurin mutants with greater anti-tumor potentials.