A novel binding site on the cryptic intervening domain is a motif-dependent regulator of O-GlcNAc transferase

Abstract

The modification of intracellular proteins with O-linked β- N -acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we have identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Utilizing an innovative proteomic peptide phage display (ProP-PD) coupled with structural, biochemical, and cellular characterizations, we discovered a novel peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs including nutrient stress response and glucose metabolism. These findings illustrate a novel mode of OGT substrate recognition and offer the first insights into the biological roles of this unique domain.

Figure 1. ProP-PD screening identified a specific OGT binding motif.

(a) Domain schematic of full-length OGT with tetratricopeptide repeat (TPR) domain in gray, N-terminal catalytic (N-Cat) domain in green, intervening domain (Int-D) in blue, and C-terminal catalytic (C-Cat) domain in yellow. The crystallization construct OGT_4.5 with only 4.5 of 13.5 TPR repeats was also shown. (b) Sequence logo of highly enriched peptides from both OGT and OGT_4.5 ProP-PD screens, aligned to the PxYx[I/L] motif. (c) Microscale thermophoresis (MST) binding assay of SMG9 peptide with OGT, n=3. (d) Competitive fluorescence polarization (FP) binding assay with fluorescently labeled 5-FAM-SMG9 peptide competing with unmodified SMG9, ZNF831, and consensus peptide 37 (CP37), for OGT binding, n=3. Error bars represent standard deviation of three replicates.

Figure 2. OGT crystal structures reveal a novel binding site in the Int-D.

(a) OGT_4.5 in complex with Int-D-binding peptides and UDP-GlcNAc. SMG9, ZNF831, and CP37 peptides are shown as cartoon in magenta, purple and blue, respectively. Protein domains in OGT_4.5 are colored as in Figure 1 a. UDP-GlcNAc is shown in orange sticks. (b) Zoom-in view at the Int-D binding site demonstrating hydrophobic interactions between SMG9 peptide (shown as sticks) and Int-D in OGT_4.5 (shown as surface). Red to white color scale represent hydrophobicity. (c) Left: surface representation of SMG9 peptide bound to OGT_4.5. Right: zoom-in view at the Int-D binding site demonstrating polar interactions between SMG9 peptide (shown as magenta sticks) and Int-D in OGT_4.5 (shown as surface) with interacting OGT residues shown in sticks. 2F_o-F_c electron density map of SMG9 peptide is shown as grey mesh and contoured at 1.0 σ. (d) Saturation fluorescence polarization (FP) binding assay of fluorescently labeled 5-FAM-SMG9 peptide with wild-type (WT) OGT (black) and mutants I734R (blue), I787E (orange), and N791A (red), n=3. (e) Competitive FP binding assay of WT SMG9 (black), mutant SMG9 Y147F (orange) and phosphorylated SMG9 pY147 (red) peptides with fluorescently labeled 5-FAM-SMG9 peptide binding to OGT, n=3. Error bars represent standard deviation of three biological replicates.

Figure 3. Int-D binding site is a regulator of protein association and O-GlcNAcylation.

(a) Co-IP of cMyc-SMG9 with Flag-OGT (W) or Flag-OGT-N791A (M) from TRex-293 cells, followed by western blot detection. (b) Co-IP of cMyc-SMG9 (W) or cMyc-SMG9-Y147F (M) with Flag-OGT from TRex-293 cells, followed by western blot detection. (c) O-GlcNAcylation detection on SMG9 (W) or SMG9-Y147F (M) from cells co-expressed with Flag-OGT (W) or Flag-OGT-N791A (M). cMyc-tagged SMG9 (WT or Y147F mutant) was immunoprecipitated from TRex-293 cell lysate by cMyc-agarose, biotinylated by GalT assay, and detected by streptavidin-HRP far western blot. All whole cell lysates have endogenous OGT knocked down. Blots are representative of at least three biological replicates.

Figure 4. Bioinformatic analysis of PxYx[I/L] motif containing proteins.

(a) O-GlcNAcylated proteins (blue circle), among the 223 PxYx[I/L] motif-containing proteins (white circle). (b) Distance of known O-GlcNAcylation sites from all PxYx[I/L] motifs (pink and blue) and phosphorylated PxYx[I/L] motifs (blue). (c) Gene Ontology terms associated with motif-containing proteins. Molecular function and protein domain (pink), posttranslational modifications (orange), biological processes (blue), cellular components (purple). Dotted line represents p-value cutoff of 0.05. (d) Overlap analysis of motif-containing proteins with reported tyrosine phosphorylation anywhere on the protein (yellow oval), O-GlcNAcylation anywhere on the protein (blue oval), and tyrosine phosphorylation on the PxYx[I/L] (orange oval).

Figure 5. OGT Int-D binding site is a regulator of nutrient stress response and lactate production.

(a) Western blot detection of global O-GlcNAcylation in TRex-293 cells overexpressing OGT (W) or OGT-N791A (M) with endogenous OGT knockdown (shOGT). (b) Western blot of O-GlcNAcylation under high (H) and low (L) nutrient conditions with OGT (W) or OGT-N791A (M) overexpression. High nutrient conditions include DMEM with 4.5 g/L glucose and 10% FBS, low nutrient conditions include DMEM with 0.45 g/L glucose and 1% FBS. (c) Western blot showing time course study of O-GlcNAcylation response to low nutrient stress with OGT (W) and OGT-N791A (M) overexpression. Cells were induced for 48 hours prior to high or low nutrient treatment, then samples were collected at 24, 32, and 48 hours of treatment. (d) Growth rate of cells overexpressing OGT (black lines) or OGT-N791A (gray lines) under high (circles) and low (triangles) nutrient conditions over 72 hours, n = 4. No significant difference was detected between cell lines in either nutrient condition. (e) Media lactate concentration from cells expressing WT OGT or N791A after 24, 48, and 72 hours. Lactate concentration, from LactateGlo assay, was normalized to cell number by CellTiter-Glo assay, and quantified by lactate standard curve. **p < 0.01, error bars represent standard deviation from n = 4 biological replicates. Western blots are representative of three biological replicates.