Figure 2. OGT crystal structures reveal a 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 1a. 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, F_o–F_c electron density map is shown as green mesh and contoured at 3.0 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. Data are presented as mean values +/− SD of three replicates.