Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension

Abstract

Human NHA2 is a poorly characterized Na(+)/H(+) antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity, and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by an extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well-known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.

Fig. 1. Building the NHA2 model-structure

(a) The location of the helices of NHA2 according to secondary structure predictions (HMMTOP and TMHMM), profile-to-profile alignment (HMAP), fold recognition (FFAS03 and INUB) and pairwise alignment with NhaA are marked in different colors according to the legends. The boundaries of the TM helices that were used for the modeling here are highlighted in grey, and the TM helix numbers are marked above. (b) The suggested TM topology of NHA2. Residues are colored according to the hydrophobicity scale of Kessel and Ben-Tal, using the color bar, with blue-through-yellow indicating hydrophilic-through-hydrophobic. The long loop connecting TM1 and TM2 was omitted for clarity. Overall, the helices are hydrophobic, but they do feature polar and titratable residues, as anticipated for a transporter.

Fig. 2. Evolutionary conservation

The evolutionary conservation profiles of NHA2, NhaA and NHE1, calculated via the ConSurf web-server (http://consurf.tau.ac.il), are shown in panels (a),(b) and (c), respectively. The intra-cellular side is facing up in all panels. The structure of NhaA and models of NHA2 and NHE1 are colored according to their conservation-grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved. Positions are colored yellow in cases in which there were insufficient data to determine the conservation scores with high statistical significance. The most variable and most conserved positions of each transporter are shown as spheres. The three proteins exhibit a highly conserved core while loops and lipid-facing residues are variable, as they should.

Fig. 3. Titratable residues in the TM4-TM11 assembly region

In all panels, TM12 was omitted for clarity and the intra-cellular side is at the top. Locations of Cα atoms of highly conserved titratable residues in the TM4-TM11 assembly region are shown as spheres. Red spheres denote aspartates and glutamates, while blue spheres indicate lysines or arginines. Ser245 and Thr462 of NHA2 are shown as green spheres. (a) The model-structure of NHA2 (light brown) is aligned to the crystal structure of NhaA (grey) and the model of NHE1 (green). The center of the TM4-TM11 assembly and flanking region is marked by a square. (b)-(d) Focus on the marked region of NHA2, NhaA and NHE1, respectively. While the assembly region of all three transporters includes conserved charged residues, each displays distinct features, possibly corresponding to functional divergence. Please refer to the main text for details.

Fig. 4. Functional analysis of NHA2 mutants predicted by homology modeling: dosage response

The salt-sensitive yeast strain AB11C was transformed with His-tagged NHA2 (WT), NHA2 mutant as indicated, or empty vector (EV). Yeasts were grown in APG medium supplemented with LiCl (Left panels), or NaCl (Right panels), and growth was determined by optical density of the culture at 600 nm (OD600) after 24 hrs (LiCl) or 48 hrs (NaCl) at 30°C. Data are the average of triplicate determinations

Fig. 5. Functional analysis of NHA2 mutants predicted by homology modeling: pH profile

Same as Fig. 4 except that LiCl concentrations was fixed at 25 mM and NaCl concentration at 300 mM. Media pH was adjusted by using phosphoric acid.

Fig. 6. Mutagenesis results in light of the evolutionary conservation analysis

In panels (a) and (b), the NHA2 model is shown as transparent ribbons, with its cytoplasmic-facing side up. Cα atoms of mutated positions are shown as spheres, and are marked by lower-case letters with their corresponding residues shown in the table. (a) Residues mutated in this study are colored by the resulting phenotype of the mutations, with red, green and yellow corresponding to non-functional, functional and partially functional. Positions were colored red even if a single mutant failed to rescue the salt-sensitive yeast in the presence of either Li⁺ or Na⁺, whereas residues were marked as partially-functional if mutations mildly lowered yeast growth in comparison to WT. (b) The model-structure is colored by conservation, as in Fig. 2a. All positions which were sensitive-to-mutation are evolutionary conserved (grades 8 and 9), while all non-sensitive positions receive significantly lower conservation scores, as anticipated.

Fig. 7. Comparison of the suggested transport mechanisms of NhaA, NHE1 and NHA2

The intracellular side is facing upwards. The schemes on the left depict the inactive conformations, which correspond to the crystal structure of NhaA and models of NHE1 and NHA2 (panels (a), (b) and (c), respectively). Indeed, pH-dependant inactive and active states were identified for NhaA and NHE, ; , and here we showed that the activity of NHA2 also depends on pH conditions (Fig. 5). Residues proposed to take part in transport and their corresponding helices are illustrated, along with the TM4-TM11 assembly. The right hand side represents the putative active conformations and the suggested transport cycles, with the residues of interest, sodium ions and protons according to the legend below. The number of transported protons matches the stoichiometry of NhaA and NHE1, and putative electrogenicity of NHA2. Since the physiological direction of transport for NHA2 is yet to be determined, its transport direction in panel (c) is for illustration purposes only. While Glu262 serves as a proton attractor in the cytoplasmic side of NHE1 (panel (b),13) and Glu252 participates in sodium binding in NhaA (panel (a),42), Glu449 may possess a similar role for NHA2 (panel (c)). The two Asp of TM5 in NhaA (panel (a)) and NHA2 (panel (c)) along with the single one of NHE1 (panel (b)) are the proposed binding sites for protons and cations in the protein core. Uniquely for NHA2, a salt bridge between Arg187 and Asp279 (panel (c)) may be formed in the active state. In the extracellular side, Ser352 and Ser362 of TM8 of NHE1 and NHA2, respectively, perhaps participate in cation binding (panels (b) and (c)). Additionally, essential Glu215 is probably situated in the ion translocation pathway of NHA2, and its precise functional role is yet to be discovered.

Fig. 8. Functional residues mapped on the structural architecture of NHA2

The cytoplasmic side is upwards. The model-structure of NHA2 is shown as transparent ribbons, whereas helices implicated in the function mechanism are marked. Helices 9 and 12, along with the extramembrane loops, were omitted for clarity. Specific residues suggested to participate in transport, illustrated in Fig. 7C, are shown as spheres and highlighted.