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.
Bibliographical noteFunding Information:
R.M. thanks Lady Davies fellowship of the Hebrew University 2019. This study was supported by a German-Israeli Project Cooperation (DIP) grant to NB-T and E.P. G.M. was funded in part by a fellowship from the Edmond J. Safra Center for Bioinformatics at Tel-Aviv University and the Madaim scholarship. N.B.-T.'s research is supported in part by the Abraham E. Kazan Chair 5 in Structural Biology, Tel Aviv University.
G.M., E.P., N.B.-T and A.F. conceptualized the project. G.M. performed the phylogenetic analyses, evolutionary conservation analyses, and homology modeling; R.M and A.R. constructed the mutants and conducted the functional assays; E.P. A.F. G.M. and N.B.-T. wrote the manuscript with input from all authors. E.P. and A.F. thank the Israel Science Foundation (ISF, grant No. 284/12 and No. 939/14, respectively).
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- NhaA antiporter
- pH-dependent Cation binding