THE CORA MG2-TYPHIMURIUM - MUTAGENESIS OF CONSERVED RESIDUES IN THE 3RD MEMBRANE DOMAIN IDENTIFIES A MG2+ PORE( TRANSPORT PROTEIN OF SALMONELLA)

The CorA transport system is the major Mg2+ influx pathway for bacteri a and the Archaea. CorA contains three C-terminal transmembrane segmen ts. No conserved charged residues are apparent within the membrane, su ggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane seg ment to identify sites involved in transport. Mutation of conserved ar omatic residues at either end of the membrane segment to alternative a romatic amino acids did not affect total cation uptake or cation affin ity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast , mutation of Tyr(292), Met(299), and Tyr(307) greatly altered the tra nsport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations ess entially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation aff inity for Mg2+, Co2+, or Ni2+ Of 10-50-fold without a significant chan ge in uptake capacity. Mutations at Tyr(307) had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A m utations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5- fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met(299), and Tyr(307) all reside on the same fa ce of the alpha-helix. Ne interpret the transport data to suggest that a hydroxyl group is important at Tyr(307), and that these three resid ues interact with Mg2+ during transport, forming part of the cation po re or channel within CorA.