title: Overexpression, Isolation, Purification, and Crystallization of NhaA.
abstract: Living cells are critically dependent on processes that regulate intracellular pH, Na(+) content, and volume. Na(+)/H(+) antiporters play a primary role in these homeostatic mechanisms. They are found in the cytoplasmic and intracellular membranes of most organisms from bacteria to humans and have long been human drug targets. NhaA, the principal Na(+)/H(+) antiporter in Escherichia coli, plays an essential role in homeostasis of Na(+) and H(+). It constitutes a paradigm for the study of its numerous prokaryotic homologs and of several human Na(+)/H(+) antiporters. The crystal structure of NhaA, determined at pH4, has provided the first structural and functional insights into the antiport mechanism and pH regulation of an Na(+)/H(+) antiporter. Remarkably, the NhaA structure revealed a new and unique fold (the "NhaA fold") that has since been observed in four additional bacterial secondary transporters. The NhaA structure has facilitated the rational interpretation of mutational data obtained in NhaA, revealing the antiporter's functional organization. Nevertheless, the crystal structure is a single snapshot, determined at acidic pH, when NhaA is downregulated; NhaA is activated at pH6.5 and reaches maximal activity at pH8.5. Therefore, it is crucial to crystallize the active conformations of NhaA. Herein, we present a procedure for determining the structure of NhaA.
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel. Electronic address: etana@vms.huji.ac.il.
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Manish
authors.initials: M
authors.lastname: Dwivedi
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003460:Crystallization / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D008958:Models, Molecular; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D006859:Hydrogen; D012964:Sodium
keywords: Cation proton antiporter super family; Membrane protein; Na(+)/H(+) antiporter; NhaA; Secondary transporter