Figure 4

Alignment of representative MHX proteins with the structural models of NCX1 and NCX_Mj. The figure shows the alignment of the MHXs of A. thaliana (At), O. sativa (Os; O.sativa_J1 is presented), P. patens (Pp; P.patens_1 is presented), and S. moellendorffii (Sm), as well as CrNCX (Cr), HsNCX1 (the Hs and N1 sequences), and NCX_Mj (Mj). To create the alignment, all MHX and NCX proteins included in Additional file 1 were first aligned by ClustalW2. Then, all proteins except those presented in Figure 4 were omitted, without changing the relative positions of the remaining proteins. This was followed by slight manual modifications and removal of gaps that appeared in all the presented proteins. For the At, Os, Pp, and Hs sequences, only residues that were totally conserved in the whole group they represented were highlighted, either in gray or as follows: red - residues conserved in all the MHXs and NCXs, including CrNCX (the four residues that are also conserved in NCX_Mj are indicated by bold, red letters in the Mj sequence); pink – a residue that was conserved in all the MHXs and NCXs except CrNCX; light green - residues conserved in all MHXs; orange - residues conserved in all the MHXs of vascular plants that differ from the corresponding residues of both P. patens paralogs; dark green - residues conserved in all the MHXs of angiosperm that differ from the corresponding residues of non-seed plants; olive-green – residues that differ between the MHXs of all monocots and all eudicots. CrNCX residues that matched the conserved sequence of all MHX or NCX proteins were highlighted in light green or yellow, respectively. The asterisks indicate residues that differ between the consensuses of all MHXs and all NCXs analysed here. Bold, underlined letters indicate regions predicted to be TMSs by the TMpred algorithm. Below the first HsNCX1 sequence (Hs), there is a duplicate sequence of this protein (N1), which presents the current structural information about NCX1. The sequence of NCX_Mj (Mj) was used to present the current structural information about this protein, and was aligned with HsNCX1 as indicated in [83] with slight modifications. The TMSs of NCX_Mj (Mj) were highlighted in light blue and the residues forming the ion binding sites, as indicated in [83], were highlighted in cyan. All the other structural information shown in this figure resulted from the study of mammalian NCX1 proteins. The experimentally determined location of NCX1 TMSs [21, 33–35] were highlighted in light blue on the N1 sequence. The position and orientation of NCX1 loops and reentrant (RE) loops were indicated below the sequences. The α1 and α2 repeat regions of NCX1, as defined in [36], were indicated by white dashes highlighted in black below the sequences. The signal peptide, XIP and CBD regions of NCX1 were indicated by pink highlighted letters in the N1 sequence. Residues highlighted in cyan or written in red letters in the N1 sequence indicate amino acids whose mutagenesis resulted in a complete loss of NCX1 activity [25, 36] or significantly altered its Ca²⁺ affinity [35], respectively. The residue numbers of the N1 sequence (indicated by bold, underlined numbers) refer to the mature protein. Regions in the large central loops of the MHXs that have a relatively high density of negatively-charged residues were indicated by red boxes.