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Structural Biology Research of Human Ap4A Hydrolase and Cloning, Expression, Purification and Preliminary X-ray Analysis of Legionella Pneumophila RpoS

Author: ChenXiaoFang
Tutor: GeHongHua
School: Anhui University
Course: Biochemistry and Molecular Biology
Keywords: Ap4A Ap4A hydrolase crystal structureL.pneumophila intracellular proliferation RpoS
CLC: R378
Type: Master's thesis
Year: 2014
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Abstract


1Structural biology research of human Ap4A hydrolaseThe dinucleotide diadenosine tetraphosphate (Ap4A) can be found in all living cells from Archeae to humans and has been proposed to be an intracellular "alarmone" both in prokaryotes and in eukaryotes. Intracellularly it affects DNA repair, RNA processing, cell division, heat shock and oxidative stress, apoptosis and transcriptional regulation. Thus the regulation of Ap4A levels must be tightly controlled.Ap4A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC3.6.1.17) is responsible for metabolizing Ap4A and is therefore involved in all the above biological processes. Recent evidence implicates that the SARS-CoV7a protein interacts with human Ap4A hydrolase and may participate in common pathways leading to cell cycle arrest and apoptosis. Ap4A hydrolase belongs to Nudix (nucleoside diphosphate linked to X) hydrolases, a superfamily of Mg2+-dependent enzymes which catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contains the conserved Nudix sequnce GX5EX7REUXEEXGU (where U represents a bulky aliphatic residue usually Ile, Leu or Val, and X represents any residue). It is characterized as cleaving the polyphosphate chain at the fourth phosphate from the bound adenosine moiety. According to phylogenetic analysis, Ap4A hydrolases were classified into two distinct groups, animal-archaeal type enzymes and plant-bacterial type enzymes. Previous studies of Ap4A structures from both groups revealed that the enzyme has the apa-sandwich architecture of a Nudix fold. However, these studies suggest that important differences exist in the binding sites between animal-archaeal type enzymes and plant-bacterial type enzymes.In this paper, we present the crystal structures of human Ap4A hydrolase E58A mutant with/without DPO. Interestingly, two sulfate ions (in wild-type protein) and one diphosphate (in E58A mutant) coordinated with some conserved residues were observed in the active site, which may have important implications in the substrate binding mode in this class of enzymes. 2Cloning, expression, purification and preliminary X-ray analysis of Legionella pneumophila RpoSLegionella pneumophila(L. pneumophila), a gram-negative bacterium, can cause fatal pneumonia after infection of alveolar tissue (Legionnaires’ disease or L. pneumophila pneumonia). Its life cycle in host cells can be divided into exponential phase (intracellular multiplication in human alveolar cells) and post-exponential phase (transmission into other host cells). The proliferation of L. pneumophila in the host cells is a programmed process and needs to sense lots of signals then respond to it, indicating the process involves a complex regulatory network. A group of genes from L. pneumophila (dot/icm:defective for organelle trafficking/intracellular multiplication) encode the Dot/Icm secretion system (Dot/Icm), homolog to the type IV secretion system. This apparatus almost involves in all the aspects of L. pneumophila parasites in the host cells, such as entry into host cells, intracellular replication, the formation of LCV (Legionella-containing vacuole), inhibition the apoptosis of host cells etc. Intracellular multiplication of L. pneumophila also requires another two "two-component systems":CpxRA (conjugation plasmid expression regulator A) and PmrA/B (polymyxin-resistant A/B) system directly regulates the transcription of the subsets genes of Dot/Icm-translocated substrates and LetAS (Legionella transmission activator/sensor) system positively regulates the expression of the genes that affect phenotype of post-exponential phase. L. pneumophila RpoS(L. pneumophila RNA polymerase sigma factor) regulates expression of the genes associated to several pathways of intracellular proliferation. In addition, the L. pneumophila RpoS has no insertion sequence in the region that homologous proteins interaction with DNA, thus lacks potential DNA binding inhibitory mechanism. A novel mechanism probably exists during the inhibition of RpoS-DNA interaction in the absence of RNAP. Therefore the structural study of L. Pneumophila RpoS may be helpful for understanding how it works in several pathways of intracellular proliferation and the mechanism of RpoS interaction with DNA in the absence of RNAP.In this work, the purification, L. pneumophila RpoS was overexpressed in E. coli. The purification, crystallization and preliminary X-ray analysis of crystals of this protein are described.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria
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