Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport |
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Authors: | Abraham Rimon Manish Dwivedi Assaf Friedler Etana Padan |
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Affiliation: | 1. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel;2. Institute of Chemistry, Faculty of Sciences, Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel |
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Abstract: | Na+/H+ antiporters have a crucial role in pH and Na+ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133. |
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Keywords: | transport protein NhaA + mutant NhaA-D133C outward-facing conformation MTSET [2-(trimethyl ammonium)ethyl] methanethiosulfonate bromide MAL-Peg MIANS (2-(4′-maleimidylanilino)-naphthalene-6-sulfonoc acid) NEM fl-NEM fluorescein 5-maleimide BTP 1,3 bis-{tris (hydroxymethyl)-methylamino} propane DDM DTT dithiothreitol ITC isothermal titration calorimetry |
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