Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters. Issue 19 (17th September 2021)

• Record Type: Journal Article
• Authors: Mondal, R.; Rimon, A.; Masrati, G.; Ben-Tal, N.; Friedler, A.; Padan, E.
• Is Part Of: Journal of molecular biology. Volume 433:Issue 19(2021)
• Publication Date: 2021-09-17
• Subjects: NhaA antiporter -- NhaA -- Cation-binding -- pH-dependent Cation binding; CPA Cation/Proton Antiporters -- TM transmembrane -- BTP Bis[tris(hydroxymethyl)methylamino]propane -- LB Luria broth -- Ni-NTA Ni-nitrilo triacetic acid-agarose -- DDM β-dodecyl-D-maltoside -- DM double mutant; 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.2021.167156
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: The molecular mechanism for NhaA pH-dependence ramifies to homologues involved in human diseases. Highlights: PH dependence of Na + /H + antiporters (CPA) underpin their role in cell pH homeostasis. Revealing the 'CPA motif' provided an insight to the pH regulation molecular mechanism. One motif's residue, Ile134 is critical for the drastic pH regulated NhaA. NhaA mutants I134T and I134T-D133S proteins exhibit pH independent Li + binding. Ile134 and Asp 133 neighboring Asp164 of the ion-binding site change its pKa. Abstract: Na + /H + antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na + and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a 'CPA motif', we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li + -binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li + binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li + more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.