Specificity and promiscuity in human glutaminase interacting protein recognition: insight from the binding of the internal and C-terminal motif

Abstract

A large number of cellular processes are mediated by protein-protein interactions, often specified by particular protein binding modules. PDZ domains make up an important class of protein-protein interaction modules that typically bind to the C-terminus of target proteins. These domains act as a scaffold where signaling molecules are linked to a multiprotein complex. Human glutaminase interacting protein (GIP), also known as tax interacting protein 1, is unique among PDZ domain-containing proteins because it is composed almost exclusively of a single PDZ domain rather than one of many domains as part of a larger protein. GIP plays pivotal roles in cellular signaling, protein scaffolding, and cancer pathways via its interaction with the C-terminus of a growing list of partner proteins. We have identified novel internal motifs that are recognized by GIP through combinatorial phage library screening. Leu and Asp residues in the consensus sequence were identified to be critical for binding to GIP through site-directed mutagenesis studies. Structure-based models of GIP bound to two different surrogate peptides determined from nuclear magnetic resonance constraints revealed that the binding pocket is flexible enough to accommodate either the smaller carboxylate (COO(-)) group of a C-terminal recognition motif or the bulkier aspartate side chain (CH(2)COO(-)) of an internal motif. The noncanonical ILGF loop in GIP moves in for the C-terminal motif but moves out for the internal recognition motifs, allowing binding to different partner proteins. One of the peptides colocalizes with GIP within human glioma cells, indicating that GIP might be a potential target for anticancer therapeutics.

FIGURE 1

GIP PDZ domain shows direct interaction with the GSSLDVTDN internal motif peptide but not with the double-substituted GSSAAVTDN peptide. (A) ¹⁵N-HSQC spectra of ¹⁵N-labeled GIP PDZ domain alone (blue) and with GSSLDVTDN peptide (magenta). (B) ¹⁵N-HSQC spectra of ¹⁵N-labeled GIP PDZ domain alone (blue) and with GSSAAVTDN peptide (magenta).

FIGURE 2

The magnitude of the amide chemical shift changes is represented in different colors on a ribbon diagram of GIP bound to the various internal motif peptides, (A) ASSSVDDMA, (B) ESSVDLLDG, (C) GSGTDLDAS, (D) GTNLDGNGD, (E) GSSLDVTDN. Red indicates Δδ> 0.2 ppm, yellow indicates 0.2 ppm > Δδ> 0.1 ppm, blue indicates 0.1 ppm >Δδ, and green indicates intermediate exchange. Selected secondary structural elements are labeled in red.

FIGURE 3

Structures of GIP-ESSVDLLDG and GIP-GSGTDLDAS complexes. (A) Ensemble of 20 lowest energy structures of the GIP-ESSVDLLDG complex. (B) D at P₊₁ forms hydrogen bonds with Leu29 & Gly30 HN and S at P₊₂ with His90. V at P₀ buries itself into a hydrophobic pocket created by Leu29, Phe31, Ile33 and Leu97. (C) Ensemble of 20 lowest energy structures of the GIP-GSGTDLDAS complex. (D) D at P₊₁ forms hydrogen bonds with Leu29 & Gly30 HN and T at P₊₂ with His90. L at P₀ buries itself into a hydrophobic pocket created by Leu29, Phe31, Ile33 and Leu97.

FIGURE 4

Localization of GIP and a GIP-binding peptide ESSVDLLDG in D54 MG human glioma cells (A) Internalization of the peptide by glioma cells. The peptide was labeled with TAMRA and is shown inside the cells as red fluorescence. (B) ESSVDLLDG peptide was localized in the cytoplasm of the glioma cell (red). (C) GIP was localized in the cytoplasm of the same cell using primary anti-GIP antibody followed by secondary antibody conjugated to Alexa 488 (green). (D) Merged image of (B) and (C) showing colocalization (indicated with arrows) of GIP and ESSVDLLDG peptide within the cell. Cell nuclei were stained with DAPI (blue).

FIGURE 5

Effect of the ESSVDLLD peptide on the cellular metabolism of D54 MG human glioma cells. The peptide was added to the cells at different concentrations ranging from 10-200 HM. Cell metabolism was determined using the MTT assay and expressed as a percentage of the mean absorbance measured in untreated control cell cultures.