Escherichia coli CorA periplasmic domain functions as a homotetramer to bind substrate.
CorA is a primary Mg2+ transporter in bacteria, which also mediates influx of Ni2+ and Co2+. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning alpha-helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-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 PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.
Wang
Shi-Zhen
SZ
Department of Biological Sciences and Biotechnology, State-Key Lab of Biomembranes and Membrane Biotechnology, Tsinghua University, Beijing 100084, China.
Chen
Yong
Y
Sun
Zhan-Hua
ZH
Zhou
Qiang
Q
Sui
Sen-Fang
SF
D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry
D027682:Cation Transport Proteins; C514433:CorA protein, E coli; D029968:Escherichia coli Proteins; D008024:Ligands; D005982:Glutathione Transferase; D013917:Thrombin; D008274:Magnesium