The CorA Mg2+ transporter is a homotetramer

Abstract

CorA is a primary Mg2+ transporter for Bacteria and Archaea. The C-terminal domain of approximately 80 amino acids forms three transmembrane (TM) segments, which suggests that CorA is a homo-oligomer. A Cys residue was added to the cytoplasmic C terminus (C317) of Salmonella enterica serovar Typhimurium CorA with or without mutation of the single periplasmic Cys191 to Ser; each mutant retained function. Oxidation of the Cys191Ser Cys317 CorA gave a dimer. Oxidation of Cys317 CorA showed a dimer plus an additional band, apparently cross-linked via both Cys317 and C191. To determine oligomer order, intact cells or purified membranes were treated with formaldehyde or carbon disulfide. Higher-molecular-mass bands formed, consistent with the presence of a tetramer. Cross-linking of the Bacillus subtilis CorA expressed in Salmonella serovar Typhimurium similarly indicated a tetramer. CorA periplasmic soluble domains from both Salmonella serovar Typhimurium and the archaeon Methanococcus jannaschii were purified and shown to retain structure. Formaldehyde treatment showed formation of a tetramer. Finally, previous mutagenesis of the CorA membrane domain identified six intramembrane residues forming an apparent pore that interacts with Mg2+ during transport. Each was mutated to Cys. In mutants carrying a single intramembrane Cys residue, spontaneous disulfide bond formation that was enhanced by oxidation with Cu(II)-1,10-phenanthroline was observed between monomers, indicating that these Mg2+-interacting residues within the membrane are very close to their cognate residue on another monomer. Thus, CorA appears to be a homotetramer with a TM segment of one monomer physically close to the same TM segment of another monomer.

FIG. 1.

Addition of a Cys residue at the C terminus of CorA allows dimer formation. Salmonella serovar Typhimurium strains MM1318 (Cys191Ser), MM1322 (Cys317), and MM1324 (Cys191Ser Cys317) and a wild-type strain (MM1442) were grown in LB broth to an OD₆₀₀ of 0.5 to 1.0, and membrane fractions were incubated with or without 10 mM tetrathionate. The samples were then run on nondenaturing PAGE before Western blotting with anti-CorA antibody and scanning into Canvas. No editing was performed except adjustment of brightness and contrast. Panel A shows the wild-type, the Cys⁻ mutant, and the double mutant strains treated with tetrathionate. Some preparations showed a small amount of the 91-kDa band before tetrathionate oxidation (see panel B); the amount of the 91-kDa species was markedly increased by oxidation with tetrathionate. Panel B shows the formation of a third band at 110 kDa in the Cys 317 mutant containing both a cytoplasmic (Cys317) and a periplasmic (Cys191) cysteine. The band was enhanced by tetrathionate oxidation, in addition to the 91- and 44-kDa bands.

FIG. 2.

Formaldehyde cross-linking indicates that CorA is a tetramer. Intact cells of E. coli DH5α, wild-type Salmonella serovar Typhimurium LT2 (MM1442), Salmonella serovar Typhimurium MM281 carrying pMAS29, a high-copy plasmid expressing CorA (MM1927), or Salmonella serovar Typhimurium MM1324, carrying the same plasmid vector expressing the Cys191Ser Cys317 CorA, were grown and treated as described in Materials and Methods. The resulting Western blot was scanned into Adobe Photoshop 5.0 and transferred into Canvas. No editing was done except some alteration of brightness and contrast. Gels from several different experiments were exposed for various times before development. Note that virtually all E. coli CorA was cross-linked by formaldehyde, while in the other three strains formaldehyde converted a large amount of monomer to higher-molecular-weight bands.

FIG. 3.

Circular dichroism spectrometry shows that purified CorA-PPD retains it structure. The purified Salmonella serovar Typhimurium CorA-PPD was used to obtain a circular dichroic spectrum on a Jasco 600 spectropolarimeter (see Materials and Methods). The negative maximum from about 209 to 220 nm is indicative of α-helical structure. Computer deconvolution of the spectrum indicates that the protein is at least 70% α-helix with no detectable β-sheet structure.

FIG. 4.

Formaldehyde treatment of purified M. jannaschii CorA-PPD indicates a homotetramer. The M. jannaschii CorA-PPD protein was purified and subjected to formaldehyde treatment as described in Materials and Methods and the legend to Fig. 2. The resulting gel was scanned into Canvas. No editing was performed except adjustment of brightness and contrast. Additional experiments at different protein or formaldehyde concentrations or different times of exposure to formaldehyde give similar results. Boiling of the samples before electrophoresis regenerated a significant amount of the monomer band as expected (data not shown). In the experiment shown, formaldehyde treatment was performed in the absence (left lane) or presence (right lane) of the selective CorA inhibitor, Co(III)hexaammine (12).

FIG. 5.

Intramembrane cysteine residues can cross-link to their cognate residue on another monomer. Strains were grown and membranes fractionated when treated with Cu(II)-1,10-phenanthroline as indicated in Materials and Methods. The gel was scanned into Canvas. No editing was performed except adjustment of brightness and contrast. Panel A shows cross-linking of the Tyr292Cys and Met299Cys mutants in TM3 of CorA. The Tyr292Cys CorA mutant cross-links spontaneously, but cross-linking is enhanced by Cu(II)-1,10-phenanthroline treatment. Panel B shows cross-linking of the Cys191Ser Tyr292Cys mutant of CorA demonstrating the absence of the higher-mass band of the dimer doublet, an indication that the doublet results from disulfide bond formation at position 292 only or at both position 292 and at position 191. Panel C shows Cu(II)-1,10-phenanthroline-mediated cross-linking at Tyr270Cys and at Ser274Cys in TM2.

FIG. 6.

Arrangements of TM domains of a tetrameric CorA. (A) A tetrameric CorA is symmetrically arranged around a putative Mg²⁺ pore with TM2 and TM3 forming the pore. TM1 is apparently peripheral to the core (23, 30). This necessarily places the TM2 of one monomer relatively far away from the TM2 of another monomer, likewise for TM3 domains. Cysteine cross-linking data show that TM3 of one monomer can cross-link with TM3 of another monomer, thus implying that they are very close. Similarly, residues in TM2 can cross-link to their cognate residue in TM2 of another monomer. This suggests that such a simple symmetrical arrangement is unlikely (see the text). (B) If TM domains are arranged as a dimer of dimers, a pseudo-twofold axis of symmetry is generated. This arrangement is known for both soluble and membrane proteins. It could satisfy the structural constraints imposed by intramembrane disulfide bond formation.