Figure 2

In silico analysis of PfCCT-specific protein segments. (A) The protein sequence alignment of the rat (RnCCT), human (HsCCT) and the two CCT repeat units of P. falciparum CCT (PfCCT_1 and PfCCT_2). Red boxes and red letters indicate the identical and similar amino acid residues within the catalytic domain, respectively. Blue box shows the Plasmodium-specific lysine-rich loop. Green, blue and yellow letters represent acidic, basic and hydrophobic residues in the membrane binding domain, respectively. The highly conserved catalytic domain is highlighted above the sequences (red) and a white line indicates the position of L5 loop. The membrane-induced amphipathic helix of RnCCT (brown box) with the determined autoinhibitory helix (red line) is shown for reference. The two, previously hypothesized N- and C-terminal membrane-induced amphipathic helix of PfCCT M domain is highlighted under the sequence with yellow boxes²⁴. Alignment was generated with ESpript 3.0⁴⁵. (B) Homology model of the active site of PfCCT with one potential representative conformation of the flexible lysine-rich loop in a close-up view. Catalytically important residues in the proximity of L5 loop are highlighted, based on the crystal structure of PfCCT (green, PDB: 4ZCS). The position of CDPCho (yellow) is shown in the active site. Blue colored line shows the position of the lysine residues on the main chain of the L5 loop (grey). (C) Helical representation of the putative membrane-induced amphipathic helices in the membrane-binding domain of the first and second repeat unit, respectively were made with HeliQuest⁴⁶. <µH> represents the hydrophobic momentum of each helix. The abundance of basic residues (blue) supports the higher affinity of membrane-binding towards negatively charged membrane surfaces, whilst a few acidic (red) and the vast majority of hydrophobic residues (grey) found in the longer m-AH-C helices supposedly have a role in the detection of local H⁺ accumulation and support the docking into the membrane leaflets⁴⁷.