The non-catalytic domains of O-GlcNAc cycling enzymes present new opportunities for function-specific control

Abstract

O-GlcNAcylation is an essential protein glycosylation governed by two O-GlcNAc cycling enzymes: O-GlcNAc transferase (OGT) installs a single sugar moiety N-acetylglucosamine (GlcNAc) on protein serine and threonine residues, and O-GlcNAcase (OGA) removes them. Aberrant O-GlcNAcylation has been implicated in various diseases. However, the large repertoire of more than 1000 O-GlcNAcylated proteins and the elusive mechanisms of OGT/OGA in substrate recognition present significant challenges in targeting the dysregulated O-GlcNAcylation for therapeutic development. Recently, emerging evidence suggested that the non-catalytic domains play critical roles in regulating the functional specificity of OGT/OGA via modulating their protein interactions and substrate recognition. Here, we discuss recent studies on the structures, mechanisms, and related tools of the OGT/OGA non-catalytic domains, highlighting new opportunities for function-specific control.

Keywords: Function-specific modulator; Non-catalytic domain; O-GlcNAc transferase (OGT); O-GlcNAcase (OGA).

Figure 1.

The enzymatic reactions and domain organizations of O-GlcNAc cycling enzymes (OGT and OGA). (a) Schematic of reversible O-GlcNAcylation catalyzed by OGT and OGA. The chemical structure of UDP-GlcNAc is also shown. (b) Domain architecture of full-length OGT (13.5 TPR repeats) and the construct of the reported crystal structure OGT_4.5. TPR, tetratricopeptide repeat. N-Cat and C-Cat, two lobes of OGT’s catalytic domain. Int-D, intervening domain. (c) Domain architecture of full-length OGA and the construct of the reported crystal structure OGA_cryst. IDR, intrinsically disordered region. pHAT, pseudo histone acetyltransferase domain.

Figure 2.

The new binding modes and modulators of OGT non-catalytic domains (TPR and Int-D). (a) The cryo-EM structure (PDB 7YEA) of full-length OGT dimer with TPR (dark blue), N-Cat and C-Cat (gray), and Int-D (yellow) highlighted in different colors. (b) The cryo-EM structure of full-length OGT dimer in complex with monomeric OGA polypeptides (PDB 7YEH). OGA’s N-terminal catalytic domain and part of the stalk domain are visible in one monomer. OGT domains are colored as the structure displayed in (a). (c) Top, the crystal structure of OGT_4.5 in complex with a motif-containing SMG9 peptide bound in the Int-D exosite (PDB 8FE7). Zoom-in view shows the hydrophobic and polar interactions between the OGT Int-D and SMG9 peptide residues. UDP-GlcNAc is shown in red surface. Bottom, the biological functions regulated by the OGT Int-D exosite. PxYxI represents the general Int-D peptide binding motif. (d) Cartoon representation of the mechanisms of OGT exosite inhibitors in modulating protein O-GlcNAcylation. (e) Dual-specificity aptamer achieves protein-specific O-GlcNAcylation in cells by simultaneously targeting the endogenous protein substrate β-catenin and OGT TPR domain.

Figure 3.

The crystal structure of OGA_cryst dimer in complex with O-GlcNAcylated p53 peptide (glycopeptide) substrate (PDB 5UN8). Zoom-in view shows the OGA substrate-binding cleft surface residues (highlighted in blue and magenta) in contact with p53 glycopeptide. The peptide part of the substrate is highlighted in orange and the GlcNAc modification is shown in red. The OGA’s catalytic domain and stalk domain are shown in pink and cyan, respectively.