Thermatoga Maritima Magnesium Transporter

Target ID:

CorA

Deposition Date:

Monday 17th October 2005

Families:

Membrane proteins

Structure type:

apo

Download Coordinates:

2BBJ

Authors:

V.V.LUNIN,E.DOBROVETSKY,G.KHUTORESKAYA,R.ZHANG,A.JOACHIMIAK,A.BOCHKAREV,M.E.MAGUIRE,A.M.EDWARDS,C.M.KOTH,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2BBJ

tabs

Structure Details

Mg2+ is essential for many biological processes and is the most abundant divalent cation in cells,
yet the mechanisms responsible for its cellular homeostasis are very poorly understood,
in significant part because of its unique chemistry.
The CorA family of Mg2+ influx proteins2-4 is widely distributed in the Eubacteria and Archaea. Physiologically they mediate Mg2+ influx, but Ni2+ and Co2+ influx leading to toxicity also occurs.
Eukaryotic homologues of this family exhibit sequence similarity almost exclusively in the membrane domain.
Yeast mitochondrial homologues appear to mediate only Mg2+ flux, and a bacterial CorA can complement yeast strains lacking the mitochondrial transporters.
Plasma membrane homologues can mediate Mg2+ transport and Al3+ resistance in yeast and plants.The structure of the T. maritima CorA magnesium transporter was determined in an apparently closed or nearly closed state.
The structure was solved by a combination of molecular replacement from a 1.85 Å resolution structure of the N-terminal two-thirds of the molecule, and non-crystallographic averaging.
The transporter is a funnel-shaped homopentamer with ten transmembrane helices, arranged as on the vertices of two concentric pentagons.
The channel is formed only by the inner group of five helices, one from each monomer and appears to be gated by the side chains of bulky hydrophobic residues.
The extracellular (periplasmic) region of each monomer is composed only of a short loop that connects the two transmembrane helices and contains the most highly conserved sequence in CorA.
The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the channel helices.
The wall of the funnel is lined by conserved rings of negatively charged and hydroxyl-bearing residues that may function as an electrostatic sink to draw Mg2+ into the cell.
The structure allows us to suggest a process by which changes in cytosolic magnesium ion concentration may be sensed by the intracellular portion of CorA and coupled to transporter closing.

Materials and Methods

TM0561 corA

PDB:2BBJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:
Tag:
Host:

Construct

Prelude:
Sequence:
Vector:modified pET15b (Novagen) vector in which the thrombin cleavage site (LVPR^GS) had been replaced with a TEV protease recognition site (ENLYFQ^G)

Growth

Medium:
Antibiotics:
Procedure:CorA was expressed in E.coli BL21 (DE3) in Luria Broth (LB) in the presence of 50 µg/mL of kanamycin and 100 µg/mL of ampicillin. Cells were grown at 37oC to an OD₆₀₀ of 0.6-0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 16oC.

Purification

Procedure
All procedures were carried out at 4ºC unless otherwise specified. The suspension was centrifuged for 30 minutes at 100,000 x g. The pellets were then solubilized in 250 mL Binding Buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 10mM imidazole, 1% n-dodecyl-?-D-maltopyranoside (DDM, Anatrace), protease inhibitors) and stirred gently for 12 hours. The sample was then centrifuged for 30 minutes at 100 000 x g, and the supernatant loaded onto a 1 x 10 cm Ni-NTA gravity column equilibrated with Binding Buffer. The column was washed with 20 column volumes of Wash Buffer (50 mM Hepes, pH 7.5, 500 mM NaCl, 5% glycerol, 35mM imidazole, 0.02% DDM). Bound protein was eluted with Elution Buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 200mM imidazole, 0.02% DDM) and dialyzed overnight against Dialysis Buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol). During dialysis, removal of the hexahistidine tag was facilitated by the addition of histidine-tagged TEV protease (Invitrogen), according to the TEV protease technical manual (Invitrogen). Digestions were monitored by SDS-polyacrylamide gel electrophoresis. The resulting proteins contained three additional residues at the N terminus (Gly-Ser-His). TEV protease and the histidine tag were separated from CorA by collecting the flow-through from a second Ni-NTA column purification, as described earlier.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For purification, the cell paste was thawed and resuspended in ice-cold Lysis Buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, protease inhibitors (Complete Protease Inhibitor Cocktail (Roche), according to the technical manual)) and lysed using a French press
Concentration:
Ligand
MassSpec:
Crystallization:Protein solutions were used immediately after purification or after storage at -78°C. Crystals were grown by the hanging drop method at 22°C, at a concentration of 2-4 mg/mL. 2 µl protein was mixed with 2 µl reservoir solution containing 20% (w/v) PEG 2000 (Fluka), 0.3M Mg(NO3)2 and 0.1M Tris pH 8.0. Needle-like crystals appeared after 3-5 days and matured to full size within 2-3 weeks.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Maguire, M. E. & Cowan, J. A. Mg2+ chemistry and biochemistry . Biometals 15, 203-210 (2002).

2. Silver, S. Active transport of magnesium in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 62, 764-771 (1969).

3. Nelson, D. L. & Kennedy, E. P. Magnesium transport in Escherichia coli. Inhibition by cobaltous ion. J. Biol. Chem. 246, 3042-3049 (1971).

4. Hmiel, S. P., Snavely, M. D., Miller, C. G. & Maguire, M. E. Magnesium transport in Salmonella typhimurium: Characterization of magnesium influx and cloning of a transport gene. J. Bacteriol. 168, 1444-1450 (1986).

5. Kehres, D. G. & Maguire, M. E. Structure, properties and regulation of magnesium transport proteins. Biometals 15, 261-270 (2002).

6. Knoop, V., Groth-Malonek, M., Gebert, M., Eifler, K. & Weyand, K. Transport of magnesium and other divalent cations: evolution of the 2-TM-GxN proteins in the MIT superfamily. Mol Genet. Genomics 1-12 (2005).

7. Hmiel, S. P., Snavely, M. D., Florer, J. B., Maguire, M. E. & Miller, C. G. Magnesium transport in Salmonella typhimurium: Genetic characterization and cloning of three magnesium transport loci. J. Bacteriol. 171, 4742-4751 (1989).

8. Kolisek, M. et al. Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria. EMBO J. 22, 1235-1244 (2003).

9. Ezaki, B., Gardner, R. C., Ezaki, Y. & Matsumoto, H. Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate aluminum stress and/or oxidative stress. Plant Physiol 122, 657-665 (2000).

10. Ezaki, B., Sivaguru, M., Ezaki, Y., Matsumoto, H. & Gardner, R. C. Acquisition of aluminum tolerance in Saccharomyces cerevisiae by expression of the BCB or NtGDI1 gene derived from plants. FEMS Microbiol Lett. 171, 81-87 (1999).

11. Li, L., Tutone, A. F., Drummond, R. S., Gardner, R. C. & Luan, S. A novel family of magnesium transport genes in Arabidopsis. Plant Cell 13, 2761-2775 (2001).

12. Schock, I. et al. A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant. Plant J. 24, 489-501 (2000).

13. Gardner, R. C. Genes for magnesium transport. Curr. Opin. Plant Biol. 6, 263-267 (2003).