Physiological, Structural, and Functional Analysis of the Paralogous Cation–Proton Antiporters of NhaP Type from Vibrio cholerae

Abstract

The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.

Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in Vibrio cholerae

“…AMLRD is widely used to inhibit the mammalian Na + /H + exchanger isoform 1 (NHE1) (Pedersen et al 2007) in cardiac diseases. Both NHE1 and Vc-NhaP2 belong to the same cation/proton antiport superfamily-1 (CPA-1) and have similarities at the protein structural level (Mourin et al 2019). Amiloride and its different analogs have also been used as anti-cancer drugs (Mihaila 2015).…”

“…The generated structure of Vc-NhaP2 has 13 Transmembrane Segments (TMSs) with TMSs V and XII being discontinuous; TMSs V and XII have extended chain region that cross each other in the middle of the membrane (Mourin et al 2018(Mourin et al , 2019) (Supplementary Figure 1).…”

“…The discontinuous region is highly flexible and crucial for the functioning of the antiporter (Mourin et al 2018(Mourin et al , 2019. The extended chain region is conserved among all the Na + /H + antiporters (present in both prokaryotes and eukaryotes) and plays crucial role in ion transport (Feig 2016, Mourin et al 2019, Padan et al 2016, Resch et al 2011. The extended chain region creates a unique electrostatic environment in the middle of the membrane which is essential for the cation binding (Padan et al 2016).…”

“…The apo-V. cholerae had much higher survival rate compared to the triple deletion mutant, Vc∆NhaP123, when challenged by HCl (pH 3.5) (Mourin et al 2017). We therefore proposed that Vc-NhaP paralogues play a critical role in the Acid Tolerance Response (ATR) of V. cholerae as it passes through the gastric acid barrier of the stomach (Mourin et al 2017(Mourin et al , 2019. In particular, after being ingested by humans V. cholerae has to pass through the low pH and high potassium (Geibel 2005;Heitzmann and Warth 2007) stomach gastric acid barrier before reaching the small intestine for colonization and secretion of the cholerae toxin (Merrel andCamilli 1999, 2000;Merrel et al 2002).…”

“…Vc-NhaP2 is the most active among the three Vc-NhaP1, 2 and 3 paralogues . Based on mutagenesis experiments and in silico analyses, we have gathered extensive information on the structural, biological, and functional properties of the Vc-NhaP2 antiporter (Mourin et al 2018(Mourin et al , 2019Schubiger et al 2017). The structural analysis of Vc-NhaP2, based on the mutagenesis data, combined with the in silico structure modelling and Molecular Dynamics (MD) Simulations yielded two important elements in the organization of Vc-NhaP2: (1) a putative cation binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, and (2) a cluster of amino acid residues near the putative cation-binding pocket determining the ion selectivity (Mourin et al 2018(Mourin et al , 2019Schubiger et al 2017).…”