Structure-function Study On Nucleotidases From Yeast And Toxin-antitoxin Systems From Escherichia Coli

The three dimensional structural study on biological macromolecules is beneficial for the understanding of their functions,which can serve as candidates targeted for drug design,gene modification,vaccine development,and even artificial construction of protein.Thus it has great potential in pharmacy,medical treatment,disease control and bio-chemical industry,etc.X-ray crystallography is a main method to study the structure and function of biological macromolecules.This thesis includes two parts:(1)yeast 5’-nucleotide enzyme.We explored the mechanism of 5’-nucleotide reaction;(2)Escherichia coli toxin-antitoxin complex.We revealed the mechanism of inhibiting toxicity and its transcriptional regulation.We used X-ray crystallography method to study their structures and functions.5’-Nucleotidases(5’-NTs)are important enzymes that can hydrolyze various nucleotides and maintain the level of nucleotides to ensure the normal functioning in cell.5’-NTs play an important role in the physiological and biochemical functions of individual growth,reproduction,heredity and mutation.HD-domain superfamily proteins can be used as 5’-nucleotidases and play a critical role in the metabolism of nucleotides and signal transduction.However,the structure and function of HD-domain 5 ’-nucleotide in eukaryotes have not been reported.In this paper,the structures and functions of 5’-NT YGK1(YGL101W)and YB92(YBR242W)containing HD-domain in yeast were studied,and the following results were obtained:We expressed and purified the recombinant proteins of YGK1 and YB92,and biochemically characterized them as 5’-nucleotidases.We determined the crystal structure of YGK1.The YGK1 structure revealed that an active center cleft formed in the middle loop region,and the active center accommodated a metal cation coordinated by HXnHDXnD motif.The experiments validated the YGK1 dimeric state in solution.Site-directed mutagenesis demonstrated that conserved amino acids including His89,Asp90 and Glu93 in the active center were essential for nucleotidase activity of YGK1.Other conserved amino acids(Glu114 and Glu145)have been found to be crucial to the catalytic activity of YGK1.These studies identified the key structural domain and catalytic residues of nucleotidase activity,explained the activity mechanism of 5’-NT,and laid the foundation for exploring its mechanisms for nucleotide metabolism and signal transduction.Abuse of antibacterial drugs in treatment of bacterial infection causes bacterial resistance increasing rapidly,which threats the public health in the world.Toxin-antitoxin(TA)is a kind of important biological macromolecule complex in bacteria and archaea,and currently the research of TA is concentrated in intestinal bacteria and pathogenic bacteria.TA can be involved in the formation of multidrug-resistant flora,which is an important factor affecting the spread of drug resistance.At the same time.TA participates in the programmed death of bacteria,regulates the formation of biofilm,and mediates the process of bacterial environmental adaptation and other important life processes.In order to fully understand the role of toxin and antitoxin,and the molecular base and interaction with specific DNA for autoregulation,we focus on the structural and functional study on HigBA and HicAB.two uncharaterized TAs in the model organisim E.coli,and conclude the following results:By analyzing the sequence of hicAB gene,the two ATG in the antitoxin HicB coding sequence could be used as the starting codon,which could produce two proposed forms(HicBS and HicBL).Accordingly,two corresponding forms of HicAB complexes were obtained through recombinant protein expression and purification:HicABS and HicABL Through the protein crystallization trial,it was found that the HicABS complex was relatively easier to crystallize.Thus the crystal of HicABS complex was obtained a1d the HicABS crystal diffraction data were collected.In the crystal structure of the HigBA complex,it displays a hetero-tetramer HigB-(HigA)2-HigB form.Each HigA is composed of all a-helices,and encompassing a HigB in the complex.HigA monomer dimerizes at their N-terminal domain through hydrophobic interaction,and then constitute into a HigB-(HigA)2-HigB form.In the complex structure,each HigA makes extensive contact with a globular HigB in the complex,but the putative active site of HigB is not occluded and exposed to solvent.However.HigB does not cause toxicity in the complex content.The reason for that is:HigB only cleaves RNA when it associates with the ribosome.Therefore,the mechanism of HigA inhibition for toxicity is:HigA sterically hinders HigB incorporation into ribosome and sequesters a ribosome-dependent ribonuclease activity in HigB.We further performed DNA and protein interaction experiments in vitro.We showed that the antitoxin protein HigA and complex higBA could bind two palindrome sequences in the-7 to-68 region of the higBA operator.The HigA C-terminal domain with a helix-turn-helix motif was the DNA binding region.These studies have showed a novel mechanism for toxin sequestion by TA complex and the TA interacton with DNA,which is of great significance to the study of the mechanism of inhibition of toxins and the auto-regulatory of TA system.