Probing the structure and function of human glutaminase-interacting protein: a possible target for drug design.
PDZ domains are one of the most ubiquitous protein-protein interaction modules found in living systems. Glutaminase interacting protein (GIP), also known as Tax interacting protein 1 (TIP-1), is a PDZ domain-containing protein, which plays pivotal roles in many aspects of cellular signaling, protein scaffolding and modulation of tumor growth. We report here the overexpression, efficient refolding, single-step purification, and biophysical characterization of recombinant human GIP with three different C-terminal target protein recognition sequence motifs by CD, fluorescence, and high-resolution solution NMR methods. It is clear from our NMR analysis that GIP contains 2 alpha-helices and 6 beta-strands. The three target protein C-terminal recognition motifs employed in our interaction studies are glutaminase, beta-catenin and FAS. This is the first report of GIP recognition of the cell surface protein FAS, which belongs to the tumor necrosis factor (TNF) receptor family and mediates cell apoptosis. The dissociation constant ( K D) values for the binding of GIP with different interacting partners as measured by fluorescence spectroscopy range from 1.66 to 2.64 microM. Significant chemical shift perturbations were observed upon titration of GIP with above three ligands as monitored by 2D {(1)H, (15)N}-HSQC NMR spectroscopy. GIP undergoes a conformational change upon ligand binding.
Banerjee
Monimoy
M
Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
Huang
Chengdong
C
Marquez
Javier
J
Mohanty
Smita
S
D001665:Binding Sites; D002942:Circular Dichroism; D015195:Drug Design; D005972:Glutaminase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D011994:Recombinant Proteins; C421249:TAX1BP3 protein, human; D005972:Glutaminase