|dc.description.abstract||PDZ domains are one of the most ubiquitous protein-protein interaction modules found in living systems. The glutaminase interacting protein (GIP), also known as the 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. Unlike other PDZ domain containing proteins, GIP contains only one PDZ domain. We detail here the over-expression, purification, and characterization of structure, function, dynamics and interactions of recombinant human GIP with different C-terminal target protein recognition sequence motifs by CD, fluorescence, and high-resolution solution NMR methods. The target protein C-terminal recognition motifs employed in our interaction studies are glutaminase L (LGA), β-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. We have also shown interaction studies of GIP with the C-terminus of Kir 2.3 and HPV16 E6. Interestingly, these ligands interact with different residues of GIP and show similar binding affinities. Dissociation constant (KD) values ranging from 1.97 µM to 3.66 µM indicate strong interactions between GIP and its binding partners. Similar to previously determined PDZ domain structures, GIP contains two α-helices and six β-strands. One of the most striking features of GIP is that, unlike other PDZ domain containing proteins, GIP shows apparent conformational change upon ligand binding. Interestingly, the backbone dynamics data of both free GIP and GIP-glutaminase C-terminus complex show that binding of the glutaminase C-terminal recognition motif results in a decrease in the order parameter (S2), indicating an increase in global flexibility of GIP in this complex.
Studies thus far show that GIP interacts with the C-terminus of its partner proteins. Using an f8-type phage displayed peptide library, we have discovered multiple peptides with novel internal sequence motifs that specifically bind to GIP. We identified S/T-X-V/L-D as the consensus GIP-binding motif and, using protein data base searches, found several cancer-related human proteins as potential GIP partners. NMR spectroscopy was used to investigate the mechanisms of novel internal motif recognition by GIP. Through mutagenesis, we found two amino acid residues within the peptides that are important for the interaction with GIP. Importantly, one of the peptides co-localizes with GIP within human glioma cells and inhibits their metabolism, indicating that GIP might be a potential target for anti-cancer therapeutic interventions.||en_US