Structural Study Of Transcriptional Regulators Lmo2088 And Lmo2131 From Listeria Monocytogenes

Insight into the crystal structure of transcriptional regulator lmo2088 from Listeria monocytogenes.Listeria monocytogenes is a foodborne gram-positive intracellular pathogen.L.monocytogenes is highly adaptable to the external environment because of its complicated network of?200 regulatory genes.These regulatory proteins have been further divided into different families.Among these regulatory families,the TetR family of transcriptional regulators plays a vital part in the regulation of different genes.The lmo2088 gene is a TetR family of transcriptional regulator and regulates the multidrug and toxic compound extrusion efflux pump encodes by a downstream gene lmo2087.This study focuses on the structure determination of the putative TetR family transcriptional regulator lmo2088 from Listeria monocytogenes.Full-length sequence encoding C-terminal 6×His-tagged lmo2088 was transformed into the pET30a expression vector using the Ndel and Xhol sites,and transformed into Escherichia coli DH5a for propagation and subsequently into BL21 and Rosetta strains of E.coli for protein expression and purification.The lmo2088 protein was purified by affinity and gel filtration chromatography.The purified and homogeneous proteins were subjected to crystallization using the vapor diffusion hanging drop method.Single crystals were obtained after optimization and used for crystal data collection.X-ray diffraction data were acquired at BL19U of the Shanghai synchrotron radiation facility.Crystal diffracted to a resolution of 1.7 A and for data processing XDS suit was used.Single-wavelength anomalous dispersion method was used for Se-Met labeled protein structure determination by Autosol-Phenix.Se-Met labeled protein structure was used as a model for native protein structure determination.Phenix-refine was used for the structure model refinement and for manual fitting the COOT program was used.Lmo2088 atomic coordinates were submitted with PDB code of 5ZTC.In this work,lmo2088 structure was determined.The structure shows that the regulatory proteins consist of two distinct domains:the DNA-binding domain at N-end carrying helix-turn-helix motif,whereas effector domain at C-end.To determine the consensus DNA binding site,we selected DNA sequences by SELEX selection and further validation was performed by isothermal titration calorimetry,fluorescence polarization,and electrophoretic mobility shift assay.We found that the transcriptional regulator lmo2088 preferably binds 25 bp y795H1 SELEX selected consensus oligo.In contrast,a randomly selected X333 DNA of an equivalent length of 25 bp shows lower binding with Imo2088.The fluorescence polarization results are consistent with that of isothermal titration calorimetry.The availability of the lmo2088 structure would be helpful in structure-based drug development.We speculate that the structure of lmo2088 from L.monocytogenes might be helpful in determination of its regulatory mechanism.Purification and crystallization of putative transcriptional regulator lmo2131 from Listeria monocytogenes.Listeria monocytogenes is ubiquitous and food-borne pathogen.L.monocytogenes bacterium is highly competent with the changing environmental conditions because of its intricate set-up of over 200 transcriptional regulators.Further,transcriptional regulators are subdivided into different families.Among these regulatory families,cAMP receptor protein/fumarate nitrate reductase regulator family of proteins performs a significant part in gene regulation.The Crp family factors act as activator of genes or group of genes.Lmo2131 is a family of protein from L.monocytogenes.To date,the structure and function of lmo2131 is unknown.The current study focuses on the structure determination of lmo2131.To determine the structure and function,overexpressed lmo2131 protein in E.coli BL21 cells was purified by affinity and gel filtration chromatography.Hanging drop method was used to crystallize the protein.Cryocooled single crystals were used for X-ray diffraction data collection.Two data sets were collected with a resolution of 1.9 A and 2.1 A respectively.To identify the consensus DNA sequence of lmo2131,a synthetic DNA library of 66 bp was screened.DNA oligonucleotide pools were sequenced and consensus sequences were identified as a target.We speculate that the understanding of function and structure will be helpful for drug designing strategies against multi-drug resistant L.monocytogenes strains.