Presence of an uncleaved signal peptide

The sequence alignment (Figure 34) and MjNhaP1 model (Figure 36, Figure 37) suggests that the additional helix (A) is in the N-terminal end. The amino acid sequence of the first helix aligns with N-terminal hydrophobic segments of several eukaryotic Na⁺/H⁺ exchangers that have been predicted to be signal peptides. Using a web based signal peptide prediction server, the sequence for this N-terminal hydrophobic segment of MjNhaP1 was analysed. Generally sequences for signal peptides typically show three distinct zones: an N-terminal region (n-region) which often contains positively charged residues, a hydrophobic region (h-region) of at least six residues and a C-terminal region (c-region) of polar uncharged residues with some conservation at the - 3 and - 1 positions relative to the cleavage site (Olof Emanuelsson1, 2007). A high score was obtained for all these three regions in the prediction, which suggests that the additional helix could be a signal peptide. The corresponding N-terminal hydrophobic segment of NHE1 from Homo sapiens was also analysed and here too the score indicated that this might also be a signal peptide.

A

B

Figure 35 Prediction of signal peptide. A. Analysis of N-terminal hydrophobic segment of MjNhaP1 (B) Analysis of N-terminal hydrophobic segment of NHE1. The figure shows score for sequences present in signal peptides. Typically, such sequences show three distinct zones: an N-terminal region (n-region, in green) which often contains positively charged residues, a hydrophobic region (h-region, in blue) of at least six residues and a C-terminal region (c-region, in cyan) of polar uncharged residues with some conservation at the - 3 and - 1 positions relative to the cleavage site. The method incorporates a prediction of cleavage sites (red line) and a signal peptide/non-signal peptide prediction based on a combination of several artificial neural networks and hidden Markov models.

3.8.2 3D model of MjNhaP1

Based on the 3D map of MjNhaP1 and using the X-ray structure of NhaA as a template, a model of MjNhaP1 was constructed.

The outer bundle helices III, V and XI of NhaA fitted well into the MjNhaP1 map after a slight shift but helix X required an extension and reorientation of the N-terminus in order for it to fit appropriately. The assignment of helices in this outer bundle was the same as those of the helices in the outer bundle of the X-ray structure of NhaA.

Although the density for the helices IV and XI appear to be continuous, these have been interpreted as disrupted/unwound helices (IVa/b and XIa/b) as observed in the X-ray structure of NhaA. These two helices had also appeared to be continuous in the NhaA 3D map (Williams, 2000). This was due to the low vertical (z) resolution of the 3D map of NhaA, which is similar to the vertical resolution of the 3D map of MjNhaP1. Therefore, it has been assumed that the half helices appear as continuous density and the disruption is not visible.

The assignment of helices to the density in the dimer interface was more difficult.

Due to the absence of a corresponding density in the 3D map of MjNhaP1, the β -hairpin of NhaA between helix II and I was omitted in the model. This omisson is further supported by the sequence alignment study, which showed that the corresponding sequence for the β -hairpin of NhaA was absent in the MjNhaP1 sequence. While the N-terminal part of helix II of NhaA could be placed at the same position in MjNhaP1, its C-terminal had to be shifted by ~10 Å to fit the map. In addition, the N-terminal part of helix IX of NhaA could be located in the same region in the 3D map of NhaP1 whereas the C-terminus had to be shifted by ~8 Å. The most drastic differences observed were in the region corresponding to helices VI, VII, and VIII of NhaA. These helices were shifted up to 15 Å in order to fit them into the MjNhaP1 map. In addition, helix VI was extended by ~10 Å and embraced the entire MjNhaP1 molecule.

After modelling the 12 NhaA helices into the MjNhaP1 map, a clear density in the proximity of helix VI, VII and VII was left, which could be interpreted as the 13th helix of MjNhaP1. By alignment of MjNhaP1 sequence with different antiporter sequences, it was evident that the additional helix is located in the N terminus of MjNhaP1 (Figure 34). To maintain consistency in helix numbering with NhaA we have called this additional helix, helix A.

A

B

Figure 36 Model 1 of MjNhaP1 fitted in to the 3D map. The helices of one monomer are represented in colours and the other monomer is represented in grey. In this model helix 0 (red) is located in the interface region and mediated tight dimer interactions.

According to the secondary structure prediction, there is a loop of approximately 7 amino acid residues connecting helix A and B. This short distance makes it likely that the position of helix A needs to be adjacent to helix B. However, there is ambiguity in the assignment of helix A. Based on this two models for MjNhaP1 were predicted: (1) In model 1, helix A is assigned to the density in the vicinity of helix B and J (Figure 36). Here, helix A is located in the interface region and appear to mediate tight dimer interactions with H and I of the second monomer. In this model helix G was assigned to the density in the periphery of the molecule (2) In model 2, Helix A is assigned to the density in the outermost periphery of the second monomer (Figure 37). At this position, helix A extends obliquely from one monomer to the other.

Figure 37 Model 2 of MjNhaP1 fitted in to the 3D map. The helices of one monomer is represented in colours and the other monomer is represented in grey. In this model helix 0 (red) is located in the periphery of the second monomer.