title: Characterization of pH-induced transitions of Entamoeba histolytica D-phosphoglycerate dehydrogenase.
abstract: 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.
authors.affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226031, India. Electronic address: mishravibhor@gmail.com.
authors.forename: Vibhor
authors.initials: V
authors.lastname: Mishra
authors.affiliation: Structural Bioinformatics Team, Division of Structural and Synthetic Biology, Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro, Yokohamo 2300045, Kanagawa, Japan.
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna, India.
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
authors.affiliation: Structural Bioinformatics Team, Division of Structural and Synthetic Biology, Center for Life Science Technologies, RIKEN, 1-7-22 Suehiro, Yokohamo 2300045, Kanagawa, Japan.
authors.forename: Kam Y J
authors.initials: KY
authors.lastname: Zhang
authors.affiliation: Department of Parasitology, National Institute of Infectious diseases, 1-23-1 Toyama, Shinjuku-Ku, Tokyo 162-8640, Japan.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Nozaki
mesh_terms: D004748:Entamoeba histolytica / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D050543:Phosphoglycerate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013816:Thermodynamics
chemical_list: D015800:Protozoan Proteins; D050543:Phosphoglycerate Dehydrogenase
keywords: Circular dichroism; Molecular dynamic simulation; Quenching; pH