HMAP alignments36, membrane topology of Nhx1 and NHE9 were predicted. Although there some gaps in the alignments of NHE1, Nhx1 and NHE9, none of these gaps are located in the predicted transmembrane helices (Figure 1A). The regions corresponding to the gaps in the alignment have no known functional roles. Evolutionarily conserved residues in both NHE9 and Nhx1 are located at the interfaces between the transmembrane segments, whereas the variable residues face the membrane lipids or are located in the extra-membrane loops. The three-dimensional models are compatible with the evolutionary conservation analyses of NHE. Consurf web-server (http://consurf.tau.ac.il/) was used to impose evolutionary conservation scores onto the 3D models. Thus, the structural modeling procedure should provide a good approximation of helix packing in the protein core, but the conformations of some of the extramembrane loops might deviate significantly from the native structure.
