Characterization of pH-induced transitions of Entamoeba histolytica D-phosphoglycerate dehydrogenase.

Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a functionally active homodimer at pH 7. Our earlier studies have shown that ionic interactions are essentially required for the oligomeric status and activity of the protein. Present study focuses on pH associated structural modulations of EhPGDH. Far-UV CD spectra showed loss in the secondary structure of the protein as a function of low pH, however, the protein was not completely unfolded even at pH 2. Energy minimized average simulated models of EhPGDH at different pH show stable secondary structure elements in the nucleotide binding domain (NBD) however, the substrate binding domain (SBD) was more sensitive toward acidic pH and completely unfolds at pH 2. The data suggest presence of partially folded/unfolded intermediate state at pH 2. Size exclusion chromatography shows that this intermediate has larger hydrodynamic radius compared with dimer (pH 7) or monomer (pH 5). The intermediate has poor tertiary organization with significantly exposed hydrophobic patches monitored by pH-dependent fluorescence spectroscopy and molecular dynamic simulations. Collectively, the results suggest that the two domains (NBD and SBD) of EhPGDH have independent pH-dependent structural transitions with stabilization of an intermediate state at pH 2.