title: Novel protein-protein interactions between Entamoeba histolyticad-phosphoglycerate dehydrogenase and phosphoserine aminotransferase.
abstract: Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein-protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10(-7) M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex.
authors.affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, MG Marg, Lucknow 226001, India. vibhor6strings@rediffmail.com
authors.forename: Vibhor
authors.initials: V
authors.lastname: Mishra
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Kumar
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Nozaki
authors.forename: Kam Y J
authors.initials: KY
authors.lastname: Zhang
authors.forename: Vinod
authors.initials: V
authors.lastname: Bhakuni
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004748:Entamoeba histolytica / Q000201:enzymology*; D006801:Humans; D056004:Molecular Dynamics Simulation; D050543:Phosphoglycerate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D050543:Phosphoglycerate Dehydrogenase; D000637:Transaminases; C022393:phosphoserine aminotransferase