期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：41
页码：E5575-E5582
DOI：10.1073/pnas.1510964112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe principal Na+/H+ antiporter of Escherichia coli (Ec-NhaA) is the best-characterized of the pH-regulated Na+/H+ exchangers that control cellular Na+ and H+ homeostasis, and the human homologues are potentially important drug targets. Identification of the essential components of NhaA is vital to understanding the function of the protein and has implications for evolution and protein design. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from the growing number of secondary transporters that share the unique Ec-NhaA structural fold. Mutants deleted of helices VI and VII, which form an -hairpin at the dimer interface, are defective in the assembly/stability of the Ec-NhaA dimer but retain significant transport activity, as well as regulatory properties. The Escherichia coli Na+/H+ antiporter (Ec-NhaA) is the best-characterized of all pH-regulated Na+/H+ exchangers that control cellular Na+ and H+ homeostasis. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from other antiporters that share the Ec-NhaA structural fold. This -hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a {beta}-sheet. Here we examine computationally and experimentally the role of the -hairpin in the stability, dimerization, transport, and pH regulation of Ec-NhaA. Evolutionary analysis (ConSurf) indicates that the VI-VII helical hairpin is much less conserved than the remaining transmembrane region. Moreover, normal mode analysis also shows that intact NhaA and a variant, deleted of the -hairpin, share similar dynamics, suggesting that the structure may be dispensable. Thus, two truncated Ec-NhaA mutants were constructed, one deleted of the -hairpin and another also lacking the {beta}-sheet. The mutants were studied at physiological pH in the membrane and in detergent micelles. The findings demonstrate that the truncated mutants retain significant activity and regulatory properties but are defective in the assembly/stability of the Ec-NhaA dimer.
关键词：transport protein ; helices truncation ; Na+/H+ antiporter/NhaA ; elastic network model ; ConSurf
