On Structure And Function Of Escherichia Coli Magnesium Transporter CorA

Mg²⁺ is the most abundant intracellular divalent cation and is required for many important cellular functions, including acting as an essential cofactor for numerous enzymatic reactions, maintaining genomic stability, modulating signal transduction, and playing a role in energy metabolism and cell proliferation. CorA is the first identified gene mediating Mg²⁺ influx and by far the most abundant prokaryotic magnesium transporter, which also mediates influx of Ni2+ and Co2+. Its homologues are widespread in archaea, fungi, yeast, plants, and mammals, comprised a large CorA transporter family. So far, the best studied CorA systems on function are from Salmonella typhimurium, Escherichia coli and on structure is from Thermotoga maritima CorA. However, those data also brought controversies such as: (1) Are there two subfamilies with different topology and oligomeric state existed in whole CorA family? (2) the properties of CorA, is it a channel or transporter? Besides, the key amino acid residues of E. coli CorA involved in transporting or ion selectivity were remaining unknown. In present work, the topology of E. coli and T. maritima CorA were determined by antibody labeling; the oligomeric state and the substrate affinity of E. coli CorA large periplasmic domain were characterized by electron microscopy and ligands dose dependent fluorescence quench method, respectively. Screening of key amino acid residues involved in transporting by site directed mutagenesis and constructing of E. coli CorA functional reconstitution platform were also included in this work as an effort to facilitate the further structure/function studies.In the second part of dissertation, the topology of E. coli and T. maritima CorA were assayed and compared by antibody labeling method. It suggested that the topology of E. coli were featured by the N- and C- termini at different side of plasma membrane, while both termini of T. maritima CorA were at the cytosol side. On the basis of previous report, we can conclud that E. coli CorA has three transmembrane segments, with its N- termini large soluble domain at periplasmic space; T. maritima CorA, as demonstrated by X-Ray structure, has only two membrane spanningα-helixes, with its large soluble domain at cytosol side. In the third part of present work, glutathione S-transferase (GST) fusion E. coli CorA periplasmic domain (CorA-PPD) was purified by GST affinity chromatography, and CorA-PPD was obtained by on-column thrombin digestion. Gel filtration indicated that purified CorA-PPD exists as a homotetramer. Single particle electron microscopy analysis of CorA-PPD and two-dimensional crystals of GST-CorA-PPD indicated that E. coli CorA-PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full length CorA and CorA-PPD also suggested that CorA-PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and CorA-PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified CorA-PPD retains its substrate binding ability as native CorA. The CorA-PPD structure observed here suggested that the pyramid-like homotetramer is the functional oligomeric state of E. coli CorA, at least the CorA-PPD.As a preliminary work for elucidating the transporting mechanism of E. coli CorA, the key amino acid residues participated in transporting were screened by deletion analysis and site-directed mutagenesis. Results showed that, among several residues subject to mutational analysis, although some residues of them did play an important role in maintaining transport activity of E. coli CorA, the tyrosine152 drew more attention. The hydroxyl group of tyrosine152, also its space arrangement, was of vital importance for performing E. coli CorA's Mg²⁺ uptake capacity. To facilitate detailed assay of E. coli CorA's transporting activity in vitro, purified E. coli CorA was functional reconstituted into proteoliposomes with unidirection. The reconstituted proteoliposomes were biochemically characterized and proved to meet the requirement for transporting activity assay. The Km of E. coli CorA to Ni2+ and Co2+ determined in present work, were consistent with that determine in vivo in previous report. Transporting assay also indicated that Ni2+ and Co2+ could enter into liposome via E. coli CorA in a "leakage" way following the chemical gradient; both transmembrane electric potential and pH gradient contributed to promote the transporting rate.