Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport. Issue 6 (16th March 2018)

• Record Type: Journal Article
• Authors: Rimon, Abraham; Dwivedi, Manish; Friedler, Assaf; Padan, Etana
• Is Part Of: Journal of molecular biology. Volume 430:Issue 6(2018)
• Page Start: 867
• Page End: 880
• Publication Date: 2018-03-16
• Subjects: transport protein -- NhaA -- Na+/H+ antiporter -- mutant NhaA-D133C -- outward-facing conformation; MTSET [2-(trimethyl ammonium)ethyl] methanethiosulfonate bromide -- MAL-Peg O-(2-maleimideoethyl)-O′-methyl-polyethylene glycol 5000′ -- MIANS (2-(4′-maleimidylanilino)-naphthalene-6-sulfonoc acid) -- NEM N-ethyl maleimide -- fl-NEM fluorescein 5-maleimide -- BTP 1, 3 bis-{tris (hydroxymethyl)-methylamino} propane -- DDM n-dodecyl-ß-d-maltopyranoside -- DTT dithiothreitol -- ITC isothermal titration calorimetry; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2018.01.014
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Highlights: Two amino acid residues Asp133 and Lys300 electrostatically balance the unique NhaA structural fold, of the Escherichi coli Na + /H + antiporter NhaA. We show that Asp133 fulfills three crucial roles in NhaA: (i) Its negative charge is critical for the stability of NhaA structure; (ii) its main chain is part of the active site; and (iii) its side chain functions as a pH-dependent gate; it changes conformation from an inward-facing conformation (at acidic pH) to an outward-open conformation (at alkaline pH) and opens the periplasm funnel. This study provides insight into the mechanisms of NhaA and its human homologs important in health and disease. Graphical Abstract: