| With the increasingly serious phenomenon of bacterial drug resistance,infection caused by drug-resistant bacteria imperils human health.Inhibitors targeting key regulators of quorum sensing system can inhibit the biofilm formation of pathogenic bacteria,and weaken the resistance of pathogenic bacteria to antibiotics,which has always been a research hotspot of antimicrobial therapy.Quorum sensing is a communication system in which bacteria use signal molecules to communicate with each other.It can help bacteria monitor the population density and adjust their growth state according to the changes of environment.In recent years,some natural quorum sensing inhibitors and synthetic analogues have been shown to attenuate bacterial diseases in animals and plants,which are expected to be used in the development of next generation antibiotics.Vibrio cholerae is the pathogen of human diarrhea disease cholera.Its pathogenicity is mainly caused by cholera toxin and pili.Studies have shown that virulence factors such as cholera toxin and pili are regulated by quorum sensing system.3,5-dimethylpyrazine-2-ol(DPO),as a negative regulatory signal molecule of Vibrio spp.,can inhibit the biofilm formation and virulence gene expression of V.cholerae through VqmA and VqmR mediated signaling pathways.VqmA is a LuxR type transcription factor receptor in the cytoplasm of V.cholerae,which plays an important role in regulating the pathogenicity of V.cholerae.In addition,VqmA is highly conserved in Vibrio spp.,and it may also be used as a drug target for other pathogenic Vibrio spp..However,without the complex structure of VqmA and DPO,it is difficult to explore the molecular mechanism of their interaction.Therefore,we hope to determine the structure of the VqmA-DPO complex,and provide a structural model for the molecular modification of DPO,so as to design more efficient antibacterial agents targeting VqmA.The main contents of this study are as follows:1.Based on the results of bioinformatics analysis,we obtained the recombinant VqmA protein by prokaryotic expression system,and determined the crystal structures of VqmA-DPO-DNA complex at a resolution of 2.51 (?) and VqmA-DPO complex at a resolution of 1.75 (?) for the first time.In addition,VqmA is the first protein in LuxR family receptor with two determined structural conformations of binding to DNA and not binding to DNA.2.Based on the crystal structure of the two complexes,we proposed that,in the ligand binding pocket,the water atoms,Phe67 and Lys101 play a key role in the recognition and binding of DPO.At the same time,the stable binding of DPO also requires the assistance of Tyr36 and Phe99 residues,thus revealing the binding mode of VqmA with DPO.3.Based on the structure of VqmA-DPO-DNA complex,we observed that the VqmA dimer interacts with ds DNA through α8 helix,and it used Lys185 and Glu188 residues to specifically recognize specific bases of DNA,thus explaining the molecular mechanism of interaction between VqmA and DNA.4.Based on structural analysis of the two complexes,we found that the binding of DNA would affect the secondary structure of the junction region between PAS and DBD domains,and for the first time observed and confirmed the existence of intermolecular disulfide bond in VqmA dimer.At the same time,we confirmed that the binding of DPO would affect the stability of the binding of VqmA and DNA by biochemical experiments.Therefore,we propose a DNA binding model that DPO and DNA assist to induce the allosteric process of VqmA junction region,which is helpful for us to understand the transcriptional regulation of LuxR family.5.Based on the binding mode of VqmA and DPO,we replaced the methyl group of C3 and C5 in DPO,and finally obtained DP-1,DP-5,DP-C5-1 and DP-C5-3according to the scoring function of molecular docking simulation software.Their predicted binding ability was significantly higher than that of DPO,which can provide a reference for the design of small molecular antibiotics against Vibrio spp..In conclusion,VqmA,as a regulatory protein in the quorum sensing system of Vibrio spp.,can regulate the formation of bacterial biofilm and the expression of virulence genes,and can be used as a protein target for the development of specific antimicrobial agents.We determined the structure of VqmA complex and designed the derivatives with higher binding affinity than DPO based on molecular docking simulation.This syudy can provide some experience for the subsequent development of antimicrobial agents based on quorum sensing,and help to carry out more efficient antibiotics research in the future. |
