Smith R L, Szegedy M A, Kucharski L M, Walker C, Wiet R M, Redpath A, Kaczmarek M T, Maguire M E
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, USA.
J Biol Chem. 1998 Oct 30;273(44):28663-9. doi: 10.1074/jbc.273.44.28663.
The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. 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 Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations 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, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.
CorA转运系统是细菌和古细菌摄取Mg2+的主要途径。CorA含有三个C端跨膜片段。膜内没有明显的保守带电残基，这表明Mg2+的摄取不涉及静电相互作用。我们对第三个跨膜片段内的保守残基进行了突变，以确定参与转运的位点。将膜片段两端的保守芳香族残基突变为其他芳香族氨基酸，并不影响总阳离子摄取或阳离子亲和力。突变为丙氨酸会大大减少摄取量，而阳离子亲和力变化不大，这意味着保守芳香族残基在将CorA的这个膜片段稳定在双层与水环境之间的界面上起到结构作用。相比之下，Tyr292、Met299和Tyr307的突变极大地改变了CorA的转运特性。Y292F、Y292S、Y292C或Y292I突变基本上消除了转运，对表达或膜插入没有影响。M299C和M299A突变体对Mg2+、Co2+或Ni2+的阳离子亲和力降低了10至50倍，摄取能力没有显著变化。Tyr307处的突变对阳离子摄取能力没有显著影响；然而，Y307F和Y307A突变对Mg2+和Co2+的亲和力降低了3至10倍，而与野生型CorA相比，对Ni2+的亲和力没有变化。相比之下，Y307S突变体对所有三种阳离子的亲和力降低了2至5倍。将第三个跨膜片段预测为α螺旋表明，Tyr292、Met299和Tyr307都位于α螺旋的同一面上。我们对转运数据的解释表明，Tyr307处的羟基很重要，并且这三个残基在转运过程中与Mg2+相互作用，形成CorA内阳离子孔或通道的一部分。
