We previously used multiple state-of-the-art approaches to construct alignments between NHE1 and NhaA36. An alignment of NHE9 and Nhx1 with NHE1 was constructed using the strategy described below, based on a higher shared sequence identity of 32 and 30%, respectively. ConSeq web server (http://conseq.bioinfo.tau.ac.il/) was used to generate an initial alignment. Additional pairwise alignment was calculated using the FFASO3 server57. Evolutionary conservation scores were calculated using the Bayesian method58, an empirical approach that uses Markov chain Monte Carlo methodology. Similar to the methodology used for developing the NHE1 fold, we used profile-to-profile alignments implemented in the FFASO3 server and Modeller59 to predict protein fold57. Transmembrane boundries of NHE1 guided the assignment of boundaries of the 12 transmembrane segments. Using an iterative process that included manually adjusting the alignments to reduce gaps in the transmembrane helices followed by Pfam, FFAS03 and 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
