Herz Katia, Rimon Abraham, Jeschke Gunnar, Padan Etana
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
J Biol Chem. 2009 Mar 6;284(10):6337-47. doi: 10.1074/jbc.M807720200. Epub 2009 Jan 7.
A structural model of the NhaA dimer showed that a beta-hairpin of each monomer combines to form a beta-sheet at the periplasmic side of the membrane. By Cys scanning the entire beta-hairpin and testing each Cys replacement for functionality and intermolecular cross-linking, we found that Gln47 and Arg49 are critical for the NhaA dimer and that K57C causes an acidic shift of 1 pH unit to the pH dependence of NhaA. Comparing the growth of the NhaA variants with the previously isolated beta-hairpin deleted mutant (Delta(P45-N58)) and the wild type validated that NhaA dimers have an advantage over monomers in growth under extreme stress conditions and unraveled that during this growth the apparent Km for Na+ of Delta(P45-N58) was increased 50-fold as compared with the wild type. Remarkably, the effect of the extreme stress on the NhaA variants is reversible. Testing the temperature stability (4-55 degrees C) of the NhaA variants in dodecyl maltoside micells showed that the mutants impaired in dimerization were much less temperature-stable than the wild type. We suggest that NhaA dimers are crucial for the stability of the antiporter under extreme stress conditions.
NhaA二聚体的结构模型显示，每个单体的一个β-发夹在膜的周质侧结合形成一个β-折叠。通过对整个β-发夹进行半胱氨酸扫描，并测试每个半胱氨酸替代物的功能和分子间交联，我们发现Gln47和Arg49对NhaA二聚体至关重要，并且K57C导致NhaA的pH依赖性向酸性方向偏移1个pH单位。将NhaA变体与先前分离的β-发夹缺失突变体（Delta(P45-N58)）和野生型的生长情况进行比较，证实了在极端应激条件下生长时，NhaA二聚体比单体具有优势，并且揭示了在此生长过程中，Delta(P45-N58)对Na+的表观Km与野生型相比增加了50倍。值得注意的是，极端应激对NhaA变体的影响是可逆的。在十二烷基麦芽糖苷胶束中测试NhaA变体的温度稳定性（4-55摄氏度）表明，二聚化受损的突变体比野生型的温度稳定性低得多。我们认为，在极端应激条件下，NhaA二聚体对于反向转运蛋白的稳定性至关重要。
