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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Curr Opin Struct Biol. 2019 Jan 30;56:97–106. doi: 10.1016/j.sbi.2018.12.003

Structural characterization of the O-GlcNAc cycling enzymes: insights into substrate recognition and catalytic mechanisms

Cassandra M Joiner [1], Hao Li [2], Jiaoyang Jiang [2],*, Suzanne Walker [1],*
PMCID: PMC6656603  NIHMSID: NIHMS1516959  PMID: 30708324

Abstract

Dysregulation of nuclear and cytoplasmic O-linked β-N-acetylglucosamine (O-GlcNAc) cycling is implicated in a range of diseases including diabetes and cancer. This modification maintains cellular homeostasis by regulating several biological processes, such as cell signaling. This highly regulated cycle is governed by two sole essential enzymes, O-GlcNAc transferase and O-GlcNAcase that add O-GlcNAc and remove it from over a thousand substrates, respectively. Until recently due to lack of structural information, the mechanism of substrate recognition has eluted researchers. Here we review recent successes in structural characterization of these enzymes and how this information has illuminated key features essential for catalysis and substrate recognition. Additionally, we highlight recent studies which have used this information to expand our understanding of substrate specificity by each enzyme.

Graphical Abstract

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Introduction

Historically, eukaryotic protein glycosylation was thought to occur exclusively in the endoplasmic reticulum and Golgi apparatus as part of the secretory pathway, which produces a vast array of diverse membrane glycoproteins. In the mid-1980’s, however, Hart and coworkers found O-linked β-N-acetylglucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins (Figure 1a) [1]. The O-GlcNAc modification is dynamic, and its addition and removal are governed by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (Figure 1b) [2–4]. Thousands of nucleocytoplasmic proteins are substrates of these O-GlcNAc-cycling enzymes. Because O-GlcNAc levels change substantially in response to nutrient availability and multiple forms of environmental stress (e.g., hypoxia, oxidative stress, thermal stress), it is thought that OGlcNAc cycling serves to maintain cell homeostasis by impacting cell signaling, gene expression, and proteostasis, among other processes [5,6]. Dysregulated O-GlcNAc abundance has been linked to several human diseases, including diabetes, cardiovascular disease, cancer, and neurodegenerative diseases, and it has been speculated that OGT and OGA may be therapeutic targets [7–10]. While the importance of O-GlcNAc cycling in metazoan physiology is by now indisputable, the functional significance of O-GlcNAc on individual substrates is extraordinarily challenging to decipher because there are so many O-GlcNAc substrates and the rules governing substrate selection are still unclear. Therefore, methods to selectively manipulate the cellular repertoire of O-GlcNAc are currently limiting. For OGT, the challenge is compounded by the recent discovery that this enzyme uses the same active site to attach O-GlcNAc and to effect another physiologically relevant modification, the cleavage of the essential cell cycle regulator, HCF-1 (Figure 1b) [11,12]. Progress in deconvoluting the functions of the O-GlcNAc cycling enzymes depends on having structural information to guide cellular experiments. A number of major advances have been made on this front in the past five years. This review will summarize key findings of structural studies on human OGT and OGA, with our apologies for the many omissions made due to space limitations. Information about structures mentioned in the text is provided in Figure 1

Figure 1: O-GlcNAc cycling is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA).

Figure 1:

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(a) Assembly of most cellular glycoproteins occur in the endoplasmic reticulum and Golgi apparatus as part of the secretory pathway. O-GlcNAc is uniquely found on nuclear and cytoplasmic proteins. (b) The O-GlcNAc cycling, the addition and removal of nuclear and cytoplasmic protein O-GlcNAc modifications, is controlled by two essential enzymes, OGT and OGA, and serves to maintain cellular homeostasis. OGT also catalyzes a second, physiological relevant protein modification, the cleavage of the transcriptional coactivator HCF-1. (c) Schematic of human OGT and constructs used for crystallography. (d) A partial list of human OGT crystal structures with different ligands. (e) Peptide sequences crystallized with OGT4.5. The TAB1 peptide was covalently fused to the N-terminus of OGT4.5. (f) Cartoon representation of human OGA isoforms and constructs for crystallography. (g) A partial list of reported human OGA crystal structures from different constructs and in complex with different ligands. The superscript numbers following PDB codes in Figure 1d and 1g represent the corresponding references.

O-GlcNAc Transferase

OGT belongs to the metal-independent GT-B superfamily of glycosyltransferases, which has been well-reviewed previously [13,14]. OGT is an essential gene encoded on the X-chromosome and it has two main regions: a long N-terminal tetratricopeptide repeat (TPR) region and a C-terminal catalytic region (Figure 1c) [15–17]. The two Rossmann-folded catalytic lobes that are characteristic of GT-B superfamily members are separated in primary sequence by a unique intervening domain of unknown function. Although there are two shorter splice variants, the TPR region of the primary, full-length OGT contains 13.5 TPRs. These are 34 amino acid helix-turn-helix motifs that typically mediate protein-protein interactions [18]. Consistent with this, removing TPRs from OGT abolishes protein glycosylation even when the active site is still functional; moreover, some cellular proteins have been shown to form stable interactions with the TPR region [17,19–22]. A crystal structure of the TPR region of OGT obtained in 2004 (PDB 1W3B; Figure 1c and 2d) shows an elongated, right-handed superhelix [23]. More recently, a structure of the TPR region containing a single point mutation distorts the superhelix, and it is speculated that this distortion alters the O-GlcNAc proteome or the OGT interactome, affecting brain development [24••].

Figure 2: OGT crystal structures provide clues to catalytic mechanisms and substrate recognition.

Figure 2:

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(a) Left panel: Cartoon representation of the structure of OGT complexes with CKII, a glycosylation substrate (yellow spheres), and UDP (magenta spheres). Middle panel: View of the active site of the OGT4.5-UDP-5SGlcNAc-CKII(A) complex showing key residues, selected diphosphate contacts and important bound water molecules. The β-phosphate of UDP-5SGlcNAc is anchored at the N-terminus of an α-helix and is stabilized by K842; a substrate-substrate interaction between the α-phosphate of UDP-5SGlcNAc and a backbone amide of CKII is proposed to assist in catalysis. The D554 side chain also plays an important role in catalysis. Right panel: Mechanism of O-GlcNAcylation highlighting the proposed role of D554 in acting as a terminal proton acceptor. (b) Left panel: Cartoon representation of OGT4.5-UDP-5SGlcNAcHCF-1rep complex shows that the cleavage region of HCF-1rep binds in the active site while the threonine-rich region binds in the TPR lumen. Middle panel: Composite structure of full-length OGT built in Pymol from PDB 4N3B and 1W3B. Right panel: The CKII(A) peptide (yellow sticks) and the cleavage region of HCF-1rep(Q) (cyan sticks) bind almost identically over UDP-5SGlcNAc (magnetic sticks) in the active site of OGT, providing structural evidence that glycosylation on glutamate is the first step in the mechanism of HCF-1 cleavage. (c) Substrates are anchored in the TPR lumen of OGT through bidentate contacts to conserved asparagines. Close-up view of contacts from asparagine side chains (magenta) to the threonine-rich region of HCF-1rep(Q) (cyan sticks; upper panel) reveals key interactions that anchor substrate in the TPR lumen; the same contacts are observed for a TAB1 peptide in the structure of an OGT4.5-TAB1 fusion protein (orange sticks; lower panel). (d) Cartoon representation of the structure of the TPR region of OGT implicated in substrate recognition. Asparagine residues in the TPR lumen are highlighted in magenta. e) Chemical mechanism of HCF-1 cleavage. Glycosylation on glutamate is followed by on-enzyme decomposition to an internal pyroglutamate, which undergoes spontaneous hydrolysis after release from OGT. The C-terminal cleavage product contains an N-terminal pyroglutamate.

The first structure of the catalytic region of human OGT was reported in 2011 (PDB 3PE4; Figure 2a) [25]. This structure, a complex with UDP and a peptide substrate (CKII), contained 4.5 TPRs. The TPRs in this structure partially overlap with the previously crystallized TPR region and a full-length model of OGT can be generated by superimposing these structures (Figure 2b, middle panel). All OGT structures obtained subsequently have used the 4.5 TPR construct as it crystallizes readily, and multiple structures of OGT complexed with substrates, substrate analogs, and products have been reported since 2011 (Figure 1d) [26–28]. Many of these are below 2.5 00C5 and include bound waters. To obtain ternary complexes that reflect how the substrates bind (i.e., pseudo-Michaelis complexes), it was necessary to use a hydrolysis-resistant analog of UDPGlcNAc in which the ring ether oxygen is replaced with sulfur (UDP-5SGlcNAc) and/or to substitute the reactive serine or threonine in the peptide substrate with alanine or aminoalanine [26]. Ternary glycopeptide product complexes were obtained simply by setting up crystals with UDP-GlcNAc or UDP-5SGlcNAc and a peptide substrate. Sulfur substitution does not alter the conformation of the substrates or products. Overall, the OGT substrate and product complexes have provided useful information about substrate preferences and reaction mechanisms.

The following picture has emerged for substrate recognition. OGT first binds UDP-GlcNAc and then the peptide substrate binds over it, making contact with the nucleotide-sugar for almost its entire length. The peptide backbone is anchored by polar contacts to OGT side chains, and there is also a polar contact between the α-phosphate of UDP-GlcNAc and the amide of the residue that becomes glycosylated, but there are almost no contacts from OGT to substrate side chains. This seemingly strange state of affairs is consistent with what is known about the lack of a strong consensus sequence for glycosylation. Some sequence preferences, such as prolines and β-branched residues in positions flanking the glycosite, have been identified from comprehensive glycosite mapping, but are insufficient to determine whether a site is glycosylated; these preferences can largely be explained by the requirement for the peptide to adopt an extended conformation over several residues in the active site [25,28,29••]

OGT complexes obtained with peptide substrates that bind in the TPR lumen of the 4.5 construct have provided important insight into how OGT substrates are selected. The first such complexes contained peptides derived from the HCF-1 cleavage region (PDBs 4N3A, 4N3B, 4N3C) (Figure 2b left panel) [30]. OGT cleaves HCF-1 within one of several, centrally-located, 26-amino acid repeats that contain a cleavage motif that binds in the active site and a threoninerich region that binds in the TPR domain [11]. The threonine-rich region is anchored in the TPR lumen by several asparagines that make bidentate hydrogen bonds to the peptide backbone (Figure 2c). Cleavage was abrogated when these asparagines were mutated to alanine [30]. These structures suggested that some glycosylation substrates may use a similar binding mode. Indeed, the structure of an OGT construct fused to a peptide derived from the glycosylation substrate TAB1 shows that the TAB1 peptide binds identically to the HCF-1 threonine-rich region in the TPR lumen (Figure 2c) [31]. Moreover, a global functional analysis has shown that a substantial fraction of OGT glycosylation substrates recognize the asparagine ladder [32••]. The evidence thus indicates that many OGT substrates are selected through extensive interactions both in the active site and to the contiguous TPR lumen. An unknown fraction of OGT substrates are thought to be directed to the active site through interactions with adaptor proteins that interaction with the TPR domain [20–22], and these adaptor proteins may also exploit the asparagine ladder for recognition. In this regard, it should be noted that the asparagine ladder lines the full length of the TPR lumen (Figure 2d) [23]. Other binding modes to OGT likely exist for both substrates and adapter proteins, and further structural and biochemical studies will be required to define these interactions.

In addition to providing substantial insight into substrate recognition, structural studies have revealed important details about mechanisms of glycosylation and HCF-1 cleavage [25,27,30]. Activation of the nucleotide-sugar is accomplished through interactions of the β-phosphate with the N-terminus of an α-helix as well as side chain contacts, including a contact with K842, an essential catalytic residue (Figure 2a middle panel). The previously mentioned contact from the α-phosphate to the amide of the glycosylated residue likely plays a crucial role in catalysis by positioning the side chain in the immediate vicinity of the anomeric carbon and providing additional activation of the leaving group [25–27]. The structures have also provided strong evidence for an electrophile migration mechanism in which the sugar rotates up and away from the leaving group and towards the nucleophile during the reaction. Surprisingly, the structures of substrate complexes did not show a proximal “catalytic base” that could serve to deprotonate the nucleophile, and two hypotheses have been proposed for how the proton is removed during the reaction. In one, an α-phosphate oxygen serves as the base [26]. In the other, the proton is translocated through two ordered water molecules to D554, a residue ~7 Å from the site of reaction (Figure 2a middle and right panel) [27]. While the issue of the catalytic base is unresolved, D554 is catalytically important for glycosylation, but not for HCF-1 cleavage, a difference consistent with its function as a base required for Ser/Thr glycosylation.

The structures of OGT substrate complexes showed that peptide backbones of glycosylation and cleavage substrates are superimposable (Figure 2b right panel). Combined with biochemical evidence consistent with a reaction mechanism involving a glutamyl ester, the structural evidence suggested that HCF-1 cleavage initiates when the invariant glutamate in the cleavage motif (Gln in Figure 2b) attacks the anomeric carbon [25,30]. No base is required in this reaction as glutamate is deprotonated at the reaction pH. Other studies have led to the mechanism for cleavage shown in Figure 2e [33••]. It has been found that aspartate-containing substrates can also be glycosylated by OGT; this results in isomerization to isoaspartate via a succinimide intermediate [34••]. Whether aspartate to isoaspartate isomerization is physiologically relevant for OGT, either as a side reaction requiring repair or as a switch mechanism for activity is not yet known. OGT can also occasionally transfer GlcNAc to cysteines in cellular proteins [35••]. OGT has thus revealed that a range of post-translational modifications (O-glycosylation, S-glycosylation, peptide backbone cleavage, and peptide backbone isomerization) are possible for a glycosyltransferase only on the identity of the glycosylated residue.

In order to decipher OGT’s cellular functions in mammals, OGT mutants lacking particular functions must be evaluated in cells. Based on structural studies, progress has already been made in identifying mutants that can affect only O-GlcNAcylation or HCF-1 cleavage; mutants lacking both functions are also available to evaluate the sufficiency of a scaffolding function for OGT. Recent work has also led to a better understanding of how the TPR region of OGT is involved in substrate recognition, which will make it possible to assess how changes to this region impact cell survival.

O-GlcNAcase

Human OGA is a multidomain protein with an N-terminal domain similar to glycoside hydrolase family 84 (GH84) enzymes, a stalk domain, a C-terminal pseudo histone acetyltransferase (HAT) domain, and several low-complexity regions (Figure 1f) [36]. A splice variant that lacks the HAT domain is less abundant and less active than OGA [37]. Although human OGA was first cloned and biochemically characterized in 2001 [36], it did not yield to structural studies until 2017. In the meantime, studies of bacterial homologs possessing a GH84 domain provided valuable insights into the hydrolytic mechanism and allowed the rational design of potent and specific OGA inhibitors, including GlcNAcstatin and thiamet-G [38–44].

In 2017, three independent research groups reported structures of human OGA, in apo form and in complex with small molecule inhibitors (Figure 1g) [45–47••]. One group also reported a series of structures of OGA complexed with different glycopeptide substrates [47••,48••], and these have begun to reveal general principles for substrate recognition. Key to obtaining the structures was the identification of stable OGA constructs for protein crystallization. Different approaches were used to generate stable OGA constructs (Figure 1f), but all maintained activity comparable to full-length OGA [45–47••]. The structures showed that the catalytic domain has the classic (β/α)8-barrel conformation characteristic of GH84 hydrolases, and also revealed an unusual dimeric structure not observed for previously studied bacterial OGA homologs (Figures 3a and 3b). The stalk domain forms a helical bundle comprising four main α-helices, three from the same OGA monomer and the fourth (labeled “arm” in Figure 3a) contributed by the sister monomer via a flexible loop. The dimer is stabilized by burial of a large surface area with formation of an extensive network of hydrogen bonds, salt bridges, and hydrophobic interactions. Residues on the dimerization interface are evolutionarily conserved in eukaryotes, and biophysical studies have confirmed that mammalian OGA exists as a stable dimer in solution [46••,47••].

Figure 3. Human OGA structures provide insights into substrate recognition and inhibitor design.

Figure 3.

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(a) OGA forms an “arm-in-arm” homodimer (PDB: 5TKE). The catalytic domain and stalk domain of one monomer are shown as ribbons in orange and marine color, respectively; the sister monomer is shown as ribbon in white color. An α-helix from one monomer (arm) forms tight binding with the sister monomer. (b) Ribbon representation of the overlay of the structures obtained from four different constructs. PDB 5TKE (green), 5UHK (cyan), and 5M7R (yellow) show nearly identical dimerization while 5UHP (magenta) shows a more relaxed conformation. (c) OGA binds inhibitors in the sugar binding pocket through strong hydrogen bonding and hydrophobic interactions. The complexes of OGA with thiamet-G (Left panel), PUGNAc and PUGNAc-imidazole (Middle panel), and VV347 (Right panel) are presented with inhibitors shown as sticks in indicated colors. The catalytic domain of one monomer and the stalk domain of the sister monomer are represented as surface in white and marine color, respectively; interacting residues from the catalytic domain and stalk domain are shown as grey and marine sticks, respectively. Hydrogen bonds and hydrophobic interactions are shown as dashed lines. (d) OGA binds glycopeptide substrates in a bi-directional yet conserved conformation. Left panel: α-crystallin B chain glycopeptide (green spheres) is bound in the substrate-binding cleft of OGA. Middle panel: Five different peptides (ribbon in indicated colors) are bound in the cleft in bidirectional but conserved conformation despite their distinct sequences (p53: QLWVDSTPPPG;α-crystallin B chain: FPTSTSLSPFYLR; TAB1: VPYSSAQS; ELK1: FWSTLSPI; Lamin B1: KLSPSPSSRVTVS. Residues with clear electron density are highlighted in bold and the O-GlcNAcylation site for each peptide is underlined). The N- or C-terminus of each peptide is highlighted in bold in corresponding color. Right panel: Glycopeptides are bound as Vshape in the substrate-binding cleft of OGA and form side-chain specific interactions with cleft surface residues. The interacting residues from the catalytic domain and stalk domain of OGA are highlighted as pink and green, respectively.

The three structures of OGA-thiamet-G complexes are superimposable and reveal active site residues that engage in tight interactions with the inhibitor which is a transition state mimic (Figure 3c left panel) [45–47••]. An ordered water molecule positioned near the carbon corresponding to the anomeric position of the sugar reveals the trajectory of nucleophilic attack for glycoside hydrolysis. The structures provide direct structural evidence to support the proposed substrate-assisted mechanism for OGA [49] and unveil a set of ancillary residues contributing to the hydrolytic mechanism. A hydrophobic pocket that affords favorable contacts to the N-ethyl substituent of thiamet-G provides a structure-based rationale for selectivity against other β-hexosaminidases. PUGNAc, a potent but more promiscuous inhibitor, cannot leverage this deep pocket to achieve specific targeting of OGA (Figure 3c middle panel) [45••]. VV347, an especially potent OGA inhibitor, makes favorably contacts with OGA surface residues (Figure 3c right panel) [46••]. Taken together, the structures of OGA complexed with these compounds provide valuable knowledge to guide future efforts in inhibitor design, both for O-GlcNAc functional investigation, and potential therapeutic applications such as in Alzheimer’s disease [10].

The OGA-glycopeptide structures provide critical insight into how OGA recognizes diverse O-GlcNAcylated proteins. Structures of a catalytically impaired OGA mutant (D175N) complexed to five distinct glycopeptide substrates have been reported (Figure 1g) and show that all of these glycopeptides are bound in the substrate-binding cleft created by the dimerization of OGA (Figure 3d) [47••,48••]. Regardless of the sequence flanking the glycosylated residue or whether that residue is Ser or Thr, the GlcNAc moieties overlap perfectly and engage in significant interactions with active site residues. This sugar binding mode explains the absolute selectivity of OGA for hydrolyzing β-O-GlcNAc substrates over either α-O-GlcNAc or β-O-GalNAc substrates. More strikingly, although all of the peptides adopt a similar “V”-shaped binding conformation near the glycosylation site, the peptide backbones can be oriented in opposite directions (Figure 3d middle panel) [48••]. Some of the binding conformations are further stabilized by intramolecular peptide interactions, and some peptide side-chain specific interactions are observed with the OGA cleft, such as the W(−3) subsite of the p53 glycopeptide with F223 and W679 (Figure 3d right panel). Mutation of these contact residues substantially impaired the binding of the p53 glycopeptide for OGA. Notably, different cleft surface residues of OGA participate in interactions with distinct glycopeptides [48••]. These five glycopeptide structures thus illuminate a general mechanism for how human OGA recognizes and processes diverse protein substrates. The conserved strong interactions between the GlcNAc moiety and OGA active site residues are critical for recognizing and anchoring glycopeptides in the substrate-binding cleft, but contacts beyond the immediate catalytic pocket can enhance the binding affinity for individual substrates and may affect hydrolysis rates. In support of this, recent proteomic analysis revealed that protein OGlcNAcylation turnover rates varied dramatically in cells, depending on both the protein and the residue modified [50]. These results, in agreement with the structural discoveries, suggest that OGA favorably removes O-GlcNAc from certain substrates during dynamic O-GlcNAc regulation.

Conclusion

The past few years have witnessed substantial progress in understanding the structural mechanisms of substrate recognition and the reactions catalyzed by the O-GlcNAc-cycling enzymes, but more work remains. In particular, future research will be needed to establish how OGT and OGA interact with protein substrates and to advance understanding of substrate specificity. Structural work that reveals new binding modes and guides the design of cellular experiments to deconvolute OGT and OGA’s functions will continue to play a crucial role in linking in vitro biochemistry to cellular function. In addition, regulatory mechanisms of OGT and OGA that limit futile O-GlcNAc cycling remain to be uncovered.

Acknowledgements

This work was supported by NIH grants R01 GM094263, R01 GM121718, and R01 GM126300.

Footnotes

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Conflicts of interest statement

Nothing declared.

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