title: 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.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4965, USA.
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Warren
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Kucharski
authors.forename: Alexander
authors.initials: A
authors.lastname: Veenstra
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.forename: Paul F
authors.initials: PF
authors.lastname: Grulich
authors.forename: Michael E
authors.initials: ME
authors.lastname: Maguire
mesh_terms: D019943:Amino Acid Substitution; D001105:Archaea / Q000201:enzymology; D001412:Bacillus subtilis / Q000201:enzymology*; D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D017017:Methanococcus / Q000201:enzymology*; D008970:Molecular Weight; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry*; D012486:Salmonella typhimurium / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D021122:Protein Subunits; D008274:Magnesium; D003545:Cysteine