Looking for the molecular basis for the formation of a tetrameric structure by native CaHIcDH, homology modeling of this protein was performed. The amino acid sequences of S. pombe and C. albicans HIcDHs show over 60% identity, so that the PDB: 3ty4 structure of the former was used as a starting template for comparative modeling of the C. albicans protein. As the native S. pombe HIcDH is a homodimer, the resulting model was also dimeric. Assuming a possibility of tetrameric structure of CaHIcDH, known previously for the T. thermophilus enzyme and suggested by the results of our MW determination, the PDB: 3asj structure of the thermophilic archeon was used as a new template in the remodeling of the dimeric model obtained in the previous stage, giving finally the model shown in Fig. 5. Based on the multiple sequence alignment of S. cerevisiae, S. pombe, C. albicans, and T. thermophilus enzymes (Fig. 2), the most divergent regions of the sequences of eukaryotic versions of the enzyme are located in the loop areas 153–158, 193–202 and 228–233 (C. albicans numbering). The second and the third of these regions form solvent-exposed loops, distant from the binding site, but the first one, namely 153–158 according to the alignment with the sequence of T. thermophilus HIcDH, may be involved in the stabilization of the enzyme quaternary structure. The homologous loop of the prokaryotic protein (131–136, T. thermophilus numbering) forms the interface between subunits of the tetramer (Fig. 5). In all known eukaryotic versions of HIcDH, this loop is of a similar length and is 5–6 residues longer than the corresponding loop in the T. thermophilus protein. However, as mentioned earlier, even among eukaryotic HIcDHs, this is a very diver-gent region. It is relatively rich in charged residues (155KKED158) in the sequence of C. albicans HIcDH, has two such residues in the sequence of S. cerevisiae HIcDH (159DK160) and none in S. pombe sequence. The charged residues present in the C. albicans HIcDH 153–158 loop may participate in intersubunit interaction. These are not shown in our model resulting from the homology modeling based on the T. thermophilus template but may be present in the real protein, where the intermolecular distances might be shorter. Moreover, Tyr154 found in the C. albicans sequence, corresponds to Tyr132 of T. thermophilus but is replaced by Val140 in the sequence of S. pombe (Fig. 2). The actual quaternary structure of SpHIcDH is dimeric, whereas both the former proteins are tetramers. It is well known that the side chain of tyrosine is often involved in the quaternary structure stabilization of multimeric proteins, due to its ability to participate in polar, hydrophobic, and cation-π interactions (Brinda & Saraswathi, 2005). In the tetramer of T. thermophilus HIcDH, the hydroxyl moiety of Tyr132 forms a hydrogen bond with Asp139′ whereas its phenyl ring interacts with the hydrophobic patch formed by side chains of Val141′ and Val126′ (Nango et al., 2011). Similar interactions are preserved in our model of the C. albicans enzyme. The hydroxyl moiety of Tyr154 is involved in a strong hydrogen bond with Glu164′, whereas its phenyl ring is facing the guanidine moiety of Arg168′ and thus can be involved in the favorable cation-π type of interactions (Fig. 5). Interestingly, an analogous tyrosine residue can be identified in the sequence of S. cerevisiae HIcDH (Tyr157, Fig. 2) suggesting a possible tetrameric quaternary structure for this enzyme as well. On the other hand, the sequence corresponding to the CaHIcDH 153–158 loop in ScHIcDH contains fewer-charged residues than that of the former.
Figure 5. Model of the tetrameric CaHIcDH (top) and close-up to the interface between subunits forming tetrameric structures of the CaHIcDH model (bottom left) and the TtHIcDH crystal structure (bottom right). Charged amino acids (Arg, Glu, Asp) proposed to be participating in the interactions are drawn as thin sticks. In the TtHIcDH model, Tyr132 and other interacting residues (Asp139′ and Val141′) are drawn as thick sticks. Respective Tyr154, Glu164′ and Arg168′ residues are shown in the CaHIcDH model. For the sake of clarity, only one set (out of four, one for each subunit of the tetramer) is shown.
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