Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins

Significance

O-GlcNAc is an abundant, reversible posttranslational modification (PTM) of nuclear and cytoplasmic proteins in animals and plants. O-GlcNAc regulates a wide range of biological processes, and aberrant O-GlcNAcylation is implicated in numerous human diseases. However, key aspects of O-GlcNAc signaling remain poorly understood. For example, it is not known whether “reader” proteins exist to recognize and bind to O-GlcNAc, as is true for many other PTMs. We used a biochemical method to identify candidate human O-GlcNAc reader proteins, and then characterized them at the biochemical and biophysical levels. Our results address a significant gap in the cell signaling field by revealing the biochemical and structural basis for the recognition of O-GlcNAc by conserved human proteins.

Abstract

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by “reader” proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein–protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification (PTM) of serines and threonines on nuclear, cytoplasmic, and mitochondrial proteins, governing diverse biological processes (1–3). In mammals, O-GlcNAc is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA), and is essential, as genetic ablation of either enzyme is lethal in mice (4–6). Aberrant O-GlcNAc cycling is also implicated in numerous human diseases, including cancer (1, 7–9), diabetes (10–12), and neurodegeneration (13–16).

Despite this broad pathophysiological significance, the molecular mechanisms of O-GlcNAc signaling are poorly understood. One prevailing hypothesis is that O-GlcNAc moieties, similar to other intracellular PTMs, may be recognized by “reader” proteins that specifically bind O-GlcNAc to effect downstream functions (17, 18). However, although some examples of O-GlcNAc-mediated protein–protein interactions are known (19–26), no general O-GlcNAc readers have been identified, and no structure of any O-GlcNAc-mediated protein–protein interaction has been reported, leaving the biochemical and biophysical basis of O-GlcNAc recognition unclear. To address this knowledge gap, we devised a screen to discover candidate mammalian O-GlcNAc reader proteins. We discovered that multiple human proteins, including 14-3-3 isoforms (17, 27, 28), bind selectively to O-GlcNAcylated substrates, and we determined structures of human 14-3-3β/α and γ bound to glycopeptides, providing atomic resolution information on an O-GlcNAc-mediated protein–protein interaction. Remarkably, the 14-3-3 O-GlcNAc-binding pocket overlaps with its well-known phosphorylated ligand binding site (17, 27, 28). Therefore, 14-3-3 proteins may serve as bifunctional readers, integrating information in the previously documented, extensive crosstalk between O-GlcNAcylation and O-phosphorylation pathways (1, 29–33).

Results

We developed a biochemical approach to test the hypothesis that O-GlcNAc is specifically recognized by mammalian reader proteins. First, we derived a consensus O-GlcNAcylated peptide sequence by aligning 802 mapped Ser-O-GlcNAc sites (34–36) (Fig. 1A) (www.phosphosite.org). We noted that a Pro-Val-Ser tripeptide observed previously in smaller datasets (37, 38) also emerged in our sequence, suggesting that this motif may be important for O-GlcNAc modification and/or recognition. We reasoned that this consensus peptide, when glycosylated, might serve as biochemical “bait” for diverse O-GlcNAc reader proteins. Human OGT efficiently glycosylated a bait peptide containing the consensus sequence and a polyethylene glycol-biotin anchor (SI Appendix, Fig. S1A) with a single O-GlcNAc on the expected serine, as determined by mass spectrometry (MS; Fig. 1B and SI Appendix, Fig. S1B). The GlcNAc-binding lectin wheat germ agglutinin was affinity-enriched via pulldown with the bait glycopeptide, compared with the unglycosylated control peptide, even in the presence of excess nonspecific protein (Fig. 1C). These results indicated that our approach could selectively capture GlcNAc-binding proteins.

Fig. 1.

A biochemical strategy to identify O-GlcNAc reader proteins. (A) Peptide logo derived from 802 mammalian serine-O-GlcNAc sites. (B) Human OGT O-GlcNAcylates the bait peptide once at the central serine. MALDI spectra of the bait peptide after mock (Left) or complete (Right) O-GlcNAcylation reaction. m/z of modified peptide corresponds to +1 GlcNAc and +1 Mg²⁺. See also SI Appendix, Fig. S1B. (C) Fluorescein-tagged wheat germ agglutinin was incubated with glycopeptide or mock-modified peptide (±O-GlcNAc), without (Left) or with (Right) 10⁵-fold excess BSA as a nonspecific protein control. Captured material was washed, eluted, and analyzed by SDS/PAGE and fluorescence scanning. (D) Nuclear and cytoplasmic 293T proteins were captured by glycopeptide pulldown and analyzed by SDS/PAGE and silver stain. (E) Summary of MS proteomics results from samples shown in D. Red circles, proteins identified in unmodified peptide pulldowns (control). Blue (nuclear) and green (cytoplasmic) circles, proteins identified in glycopeptide pulldowns. Similar results were obtained with HT1080 and Jurkat cells. See also SI Appendix, Fig. S1C. (F) Glycopeptide pulldowns from 293T extracts were analyzed by IB. Tubulin (not enriched in any MS sample) is a negative control.

Using the bait glycopeptide and MS proteomics, we affinity-captured and identified dozens of endogenous nuclear and cytoplasmic proteins that selectively bound the O-GlcNAcylated probe in extracts from three different human cell lines (Fig. 1 D and E and SI Appendix, Fig. S1C). Then, we inspected our MS datasets for the proteins most enriched by the O-GlcNAcylated bait in at least two cell types (SI Appendix, Fig. S1C). One protein meeting these criteria was importin-β1, which mediates nuclear cargo trafficking (39) and was previously shown to interact directly with O-GlcNAc moieties on nuclear pore proteins in human cells (40). We confirmed by immunoblot (IB) that importin-β1 and its homolog importin-5 were specifically enriched by the bait glycopeptide (Fig. 1F). Interestingly, several other proteins enriched in our experiments exist as dimers or higher-order oligomers, possibly indicating that avidity effects from the densely glycoprotein-decorated beads contributed to their successful O-GlcNAc-specific purification (SI Appendix, Fig. S1C). These results demonstrated that our method can identify authentic human O-GlcNAc-interacting proteins.

We selected three other glycopeptide-enriched proteins for further study: α-enolase, ErbB3-binding protein (EBP1), and 14-3-3 (SI Appendix, Fig. S1C). α-enolase is a glycolytic enzyme (41), EBP1 inhibits cell growth and proliferation induced by the receptor tyrosine kinase ErbB3 (42), and 14-3-3 proteins bind phosphoserine/threonine moieties to govern numerous processes, including growth factor signaling, mitotic exit, and apoptosis (17, 27, 28). We verified that the bait glycopeptide specifically enriched both endogenous (Fig. 2A) and recombinant-purified (Fig. 2B) forms of human α-enolase, EBP1, and 14-3-3β/α and γ (representative paralogs), demonstrating that they bind O-GlcNAc directly, and not through bridging proteins. We confirmed this conclusion using fluorescence anisotropy (FA), which revealed saturable binding of all three proteins to the glycopeptide, but not to the unglycosylated peptide (Fig. 2 C and D and SI Appendix, Fig. S2). Extending these observations, we found that human α-enolase, EBP1, and 14-3-3 also directly bound a consensus glycopeptide derived from 676 mapped threonine-O-GlcNAc sites (34–36) (SI Appendix, Fig. S3) (www.phosphosite.org), and that the murine orthologs of all three proteins were also specifically affinity-enriched by bait glycopeptides (SI Appendix, Fig. S4). Together, these results demonstrate that mammalian α-enolase, EBP1, and 14-3-3 share evolutionarily conserved O-GlcNAc-binding properties.

Fig. 2.

Human protein binds O-GlcNAc directly and selectively. (A) Glycopeptide pulldowns from 293T extracts were analyzed by IB. Tubulin is a negative control. (B) Recombinant-purified proteins were analyzed by glycopeptide pulldown and IB. Protein phosphatase-1 (PP1) (not enriched in any MS sample) is a negative control. (C) Fluorescein-labeled bait peptide was mock- (blue) or O-GlcNAc-modified (red), and binding to 14-3-3γ was analyzed by FA. Representative traces from triplicate experiment are shown. mP, millipolarization. (D) Dissociation constants for glycopeptide interactions with α-enolase, EBP1, and 14-3-3γ, determined by FA.

To determine whether α-enolase, EBP1, and 14-3-3 bind O-GlcNAc moieties in vivo, we created expression constructs encoding mCherry fused to the Ser-O-GlcNAc bait sequence and a myc epitope. Endogenous 14-3-3, α-enolase, and EBP1 coimmunoprecipitated (IP-ed) with transfected mCherry-bait, but not with a serine→alanine “mutant” bait (Fig. 3A). Moreover, a specific small molecule inhibitor of OGT (5SGlcNAc) (43) greatly reduced the interaction between candidate reader proteins and mCherry-bait, whereas an OGA inhibitor (Thiamet-G) (44) had little effect (Fig. 3B). These results indicate that constitutive O-GlcNAcylation of the bait peptide sequence is efficient, and that endogenous α-enolase, EBP1, and 14-3-3 can bind O-GlcNAc moieties in human cells.

Fig. 3.

Candidate reader proteins bind O-GlcNAcylated substrates in human cells. (A) 293T cells were transfected with mCherry, mCherry-Ser-bait-myc, or mCherry-Ala-bait-myc. Lysates were analyzed by anti-myc IP and IB. (B) 293T cells were transfected as in A, incubated 24 h, and treated with vehicle, 50 µM 5SGlcNAc (OGT inhibitor), or 50 µM Thiamet-G (OGA inhibitor) for 6 h. Lysates were analyzed by anti-myc IP and IB. The EBP1 and pan-14-3-3 signal in each IP lane was quantified and expressed as a percentage of the signal from the corresponding input sample (noted directly beneath each IB). (C) AGX1(F383G)-expressing HeLa cells (40) were treated with GlcNDAz precursor and ultraviolet (or not, negative control). Lysates were analyzed by anti-O-GlcNAc IP, SDS/PAGE, and silver stain. Gel slices of defined MW ranges were analyzed by spectral index quantitation MS proteomics (74). Graph depicts selected proteins identified in the 100–200-kDa gel region and enriched more than fivefold in the +UV sample versus the −UV (control) sample. Y-axis indicates protein abundance, as quantified by mean ion current (MIC). X-axis indicates the ratio (fold-enrichment) of each protein in the +UV/−UV samples. Proteins identified in the +UV sample but absent from the −UV control are denoted as ∞ enrichment. Axis breaks indicate scale changes only. See also SI Appendix, Fig. S5. (D) AGX1(F383G)-expressing 293T cells were transfected with vector or HA-14-3-3β/α or γ, treated as indicated, and UV-irradiated. Lysates were analyzed by anti-HA IB. Squares, uncrosslinked 14-3-3. Arrows, 14-3-3 crosslinks (i.e., O-GlcNAc-mediated interactions).

To determine whether candidate reader proteins interact with native OGT substrates in living cells, we used a chemical biology strategy (40) to covalently capture endogenous O-GlcNAc-mediated protein–protein interactions. Briefly, in this approach, human cells are metabolically labeled with a diazirine-bearing GlcNAc analog (GlcNDAz), which they convert to a nucleotide-sugar species used by OGT to modify its natural substrates (40). Short UV treatment of GlcNDAz-labeled cells covalently crosslinks proteins within ∼2–4 Å of the O-GlcNDAz moiety, capturing direct binding partners but not nonspecific proteins (25, 26, 40). We performed GlcNDAz crosslinking on human cells, IP-ed lysates with an anti-O-GlcNAc antibody, separated IP-ed proteins by SDS/PAGE, and used quantitative proteomics to identify proteins within gel slices of defined molecular weight (MW) ranges (SI Appendix, Fig. S5). Then, we inspected the results for proteins detected in a UV-dependent manner above their predicted MWs, indicating O-GlcNDAz-dependent crosslinking. As expected, we observed UV-specific crosslinking in the 100–200-kDa range of both known O-GlcNAcylated proteins, such as nucleoporins and OGT itself (45–47), and known O-GlcNAc-interacting proteins, such as importins (Fig. 3C) (40). Notably, we detected 14-3-3β/α, γ, ε, and ζ/δ (MW < 30 kDa) in the 100–200-kDa gel region exclusively in the +UV sample (Fig. 3C). Similarly, α-enolase (MW 47 kDa) was enriched more than fivefold in the region above 200 kDa (SI Appendix, Fig. S5B). IBs confirmed the GlcNDAz-specific crosslinking of 14-3-3β/α and γ to discrete endogenous human proteins (Fig. 3D). Together, these results indicate that candidate reader proteins bind directly to O-GlcNAc moieties in vivo.

Elegant studies of OGA–glycopeptide cocomplexes have provided mechanistic insight into O-GlcNAc hydrolysis (48, 49). However, no structure has been reported for an O-GlcNAc-mediated protein–protein interaction, leaving the biophysical basis of O-GlcNAc recognition unknown. To address this question, we focused on 14-3-3 proteins because of their role in cell signaling through PTMs (17, 27, 50). O-GlcNAc binding is a conserved property of human 14-3-3 isoforms, because glycopeptide pulldowns specifically enriched all family members, except σ, from mammalian cell extracts (Figs. 2A and 4A and SI Appendix, Fig. S1C). Moreover, glycopeptide pulldowns (Fig. 4B) and FA (Fig. 4C and SI Appendix, Fig. S6) with recombinant-purified protein confirmed the direct and selective binding of two additional 14-3-3 isoforms to O-GlcNAc, with no saturable binding to the unglycosylated peptide detected. The affinities of 14-3-3 isoforms for the bait glycopeptide (Fig. 2D and SI Appendix, Fig. S6), although moderate, are comparable to those reported previously for some phosphopeptide ligands from physiological binding partners (51, 52). To further test the potential physiological relevance of the affinities we observed for 14-3-3 and model glycopeptides, we synthesized previously validated 14-3-3-binding phosphopeptides from the cystic fibrosis transmembrane conductance regulator and the transcription factor Snail (51, 52) (SI Appendix, Fig. S1A) and examined them under identical FA assay conditions. Consistent with prior reports (51, 52), we observed weak binding with both cystic fibrosis transmembrane conductance regulator and Snail ligands, with affinities lower than those we observed for the model glycopeptides (SI Appendix, Fig. S7), demonstrating that the 14-3-3/glycopeptide affinities we observe are on par with some physiological 14-3-3/phosphopeptide interactions. By analogy to prior studies comparing phosphopeptides and full-length phosphoproteins (53), we expect that 14-3-3 isoforms likely bind significantly tighter to native, full-length glycosylated partner proteins than they do to our consensus glycopeptides, which were designed to capture a variety of reader proteins, and hence were not selected or optimized for 14-3-3 binding in particular (Fig. 1A and SI Appendix, Fig. S3A).

Fig. 4.

Structural basis of O-GlcNAc recognition by 14-3-3 proteins. (A) 293T extracts were analyzed by glycopeptide pulldown and IB. (B and C) Recombinant-purified 14-3-3 proteins were analyzed by glycopeptide pulldown and IB (B) or FA (C). (D and E) Structures of 14-3-3γ (cyan) (D) or 14-3-3β/α (rose) (E) bound to glycopeptide (yellow peptide, green O-GlcNAc). Lines indicate protein-glycopeptide hydrogen bonds (Left), and blue mesh indicates the initial Fo-Fc map, calculated before glycopeptide addition, contoured at 2.9 σ (14-3-3γ) or 2.5 σ (14-3-3β/α) (Right). (F) Overlay of 14-3-3γ-glycopeptide (from D) and 14-3-3γ-phosphopeptide from tyrosine hydroxylase (magenta protein, red phosphopeptide; PDB 4J6S) (75). O-GlcNAc and O-phosphate moieties are highlighted by transparent surfaces. (G) Recombinant-purified wild-type or R57E, Y133E, N178Y, or V181W mutant 14-3-3γ was analyzed by glycopeptide pulldown and IB. (H) 14-3-3γ R57E binding to glycopeptide was analyzed by FA. (I) Overlays of structures of 14-3-3γ (cyan), 14-3-3β/α (green), and 14-3-3γ R57E (magenta), displaying nearly identical modes of glycopeptide binding.

Next, to elucidate the biophysical basis of O-GlcNAc binding by 14-3-3 paralogs, we obtained X-ray crystal structures of serine and threonine bait glycopeptides bound to 14-3-3β/α or γ (Fig. 4 D and E and SI Appendix, Figs. S8–10). Strikingly, the glycopeptides occupy the same amphipathic groove of 14-3-3, where phosphorylated ligands bind (54). In addition, to protein/peptide backbone contacts, 14-3-3 makes 10 hydrogen bonds with the glycan (Fig. 4 D and E), revealing the biophysical basis for O-GlcNAc-selective binding (Figs. 1–3). Moreover, the glycopeptide and protein conformations were nearly identical in the 14-3-3β/α and γ cocomplexes (Fig. 4 D, E, and I and SI Appendix, Fig. S9), providing a structural explanation for the conserved O-GlcNAc binding property of 14-3-3 isoforms (Fig. 4A). Remarkably, the bound O-GlcNAc moiety is essentially superimposable on the O-phosphate group of prior phosphopeptide-bound structures of 14-3-3, with similar residues contacting both PTMs, despite their considerable steric and electronic differences (Fig. 4F).

We next used our structures to design mutations that uncouple the O-GlcNAc and O-phosphate binding properties of 14-3-3. Mutating R57, R132, or Y133 (14-3-3γ numbering) to glutamate is known to abolish phospho-ligand binding because of electrostatic repulsion (55, 56). We reasoned that these mutants might still bind uncharged O-GlcNAc moieties. Indeed, the R57E, R132E, and Y133E 14-3-3γ mutants selectively bound glycopeptides in pulldown and FA experiments (Fig. 4 G and H and SI Appendix, Fig. S6) comparably to wild type (Fig. 2). Furthermore, we obtained the structure of a 14-3-3γ R57E-glycopeptide complex, which confirmed that O-GlcNAc binding is preserved without significant conformational changes (Fig. 4I). In a reciprocal experiment, we created N178Y and V181W mutants, which we predicted would not bind O-GlcNAc, hypothesizing that the bulkier tyrosine or tryptophan residue would sterically disrupt the key contacts made by N178 and V181 to the glycopeptide (Fig. 4 D and E). Indeed, both N178Y and V181W 14-3-3γ failed to bind O-GlcNAcylated ligands in a pulldown assay (Fig. 4G). Together, these results provide biochemical and structural insights into selective, O-GlcNAc-mediated protein–protein interactions, and reveal the biophysical basis of glycan binding by 14-3-3 proteins, a property distinct and separable from O-phosphate binding.

Finally, we leveraged our structural and mutagenesis data to further investigate the relationship between 14-3-3 and O-GlcNAc signaling in human cells. Consistent with an earlier report (57), we found that expressed wild-type 14-3-3γ was broadly distributed throughout the nucleus and cytoplasm, whereas the R57E and Y133E 14-3-3γ mutants, which bind O-GlcNAc (Fig. 4 G–I and SI Appendix, Fig. S6) but not O-phosphate (55, 56), localize specifically to the nucleus (SI Appendix, Fig. S11). Moreover, both mutants were dispersed throughout the cell on treatment with either of two structurally unrelated small molecule OGT inhibitors (SI Appendix, Fig. S11), suggesting that their localization depends on endogenous glycosylated (but not phosphorylated) nuclear ligands. These data further support a role for 14-3-3 as O-GlcNAc reader proteins in live human cells.

Discussion

Despite their importance, the biochemical mechanisms of O-GlcNAc signaling remain incompletely understood. To address this knowledge gap, we tested the hypothesis that the O-GlcNAc modification is specifically recognized by mammalian reader proteins. Several pioneering studies from the Lefebvre group previously reported that Hsp70 family chaperones bind GlcNAc-agarose resin and associate with O-GlcNAcylated mammalian proteins in a nutrient- and stress-responsive manner (58–62). However, whether Hsp70 chaperones or other human proteins serve as direct and specific O-GlcNAc-binding readers remained unclear.

Our results demonstrate that diverse human proteins bind O-GlcNAcylated substrates directly and selectively. For example, α-enolase binds O-GlcNAcylated substrates (Figs. 1–3) and is itself O-GlcNAcylated (8), indicating that glycosylation may govern glycolysis through regulated protein–protein interactions. In support of this possibility, our glycopeptide pulldowns enriched several glycolytic and pentose phosphate pathway enzymes (SI Appendix, Fig. S1C), consistent with the well-documented role of O-GlcNAc signaling in nutrient sensing (1, 2). Similarly, EBP1 binds O-GlcNAcylated ligands (Figs. 1–3) and interacts with known OGT substrates, including the transcriptional regulators E2F1 and Sin3A (63–65), suggesting that O-GlcNAcylation may influence ErbB3 signaling through regulated multiprotein complexes of EBP1. The structural basis and functional consequences of O-GlcNAc binding by α-enolase and EBP1 are currently under investigation.

The 14-3-3 isoforms, also identified in our screen as O-GlcNAc binders, play central roles in many physiological processes, including mitogenic signaling, cell cycle progression, and cell death (17, 27, 28). Our biophysical studies with 14-3-3 provide structures of O-GlcNAc-mediated protein–protein interaction, lending insight into the signaling mechanisms of 14-3-3 and O-GlcNAc alike. On the basis of these results, we suggest that 14-3-3 proteins are well-suited to serve as general O-GlcNAc readers. Our structures reveal that the amphipathic groove of 14-3-3 is especially well equipped to bind the Pro-Val-Ser/Thr motif (Fig. 1A and SI Appendix, Fig. S3) present in hundreds of natural OGT substrates (37, 38). Specifically, the proline in this glycopeptide motif occupies a shallow hydrophobic pocket of 14-3-3 that likely would not tolerate large or polar side chains (Fig. 4 and SI Appendix, Fig. S9). Similarly, the valine, although surface exposed, lies near the conserved L225 and the Cβ atoms of D228 and N229 (14-3-3γ numbering), making several favorable contacts (Fig. 4 and SI Appendix, Fig. S9). Importantly, specific 14-3-3 residues, (e.g., D129, N178, and E185, 14-3-3γ numbering) participate in O-GlcNAc binding but not O-phosphate binding (Fig. 4 and SI Appendix, Fig. S9), consistent with our observation that the O-GlcNAc and O-phosphate binding properties of 14-3-3 isoforms are genetically separable (Fig. 4 G–I and SI Appendix, Fig. S9) (55, 56). Our results also demonstrate that multiple 14-3-3 isoforms from different mammalian species bind Ser- and Thr-O-GlcNAc moieties specifically and directly (Figs. 2–4 and SI Appendix, Figs. S2–S4), likely through nearly identical biophysical contacts (Fig. 4I and SI Appendix, Fig. S9). Therefore, the amphipathic groove of mammalian 14-3-3 isoforms is an evolutionarily conserved O-GlcNAc-binding protein module, in addition to its well-established O-phosphate-binding role.

O-GlcNAc and O-phosphate often compete for identical or nearby residues on numerous substrates, producing a complex functional interplay between these PTMs (1, 29–33). Our results raise the possibility that 14-3-3 isoforms act as bifunctional reader proteins of specific O-GlcNAcylated or phosphorylated ligands. Moreover, because 14-3-3 isoforms homo- and heterodimerize (17, 27, 28), the two amphipathic grooves of a single 14-3-3 dimer could conceivably bind one O-GlcNAc and one O-phosphate, respectively, when interacting with the many proteins known to be both phosphorylated and glycosylated (1, 29–33). Indeed, some native binding partners of 14-3-3 are phosphorylated at multiple sites, providing both high- and lower-affinity phospholigands for binding 14-3-3 dimers, permitting combinatorial tuning of cell signaling through multiple PTMs on a single protein (52, 66, 67). We speculate that O-GlcNAc moieties may similarly serve as high- or lower-affinity 14-3-3 binding sites, perhaps in combination with phosphorylation sites, on endogenous human proteins. Testing these hypotheses will be an important goal for future studies.

The natural O-GlcNAcylated binding partners of 14-3-3 proteins remain to be identified. However, our proteomics results revealed several known endogenous nuclear glycoproteins in the same GlcNDAz-crosslinked, high-molecular-weight complexes as endogenous 14-3-3 (Fig. 3C and SI Appendix, Fig. S5). Importantly, the chemical mechanism and short crosslinking radius of the GlcNDAz reagent ensure that these in vivo interactions are mediated by direct binding to glycans (40). These data suggest that 14-3-3 may exploit high O-GlcNAc ligand avidity to bind directly to heavily glycosylated nucleoporins or other nuclear OGT substrates (68–70). Consistent with this hypothesis, our microscopy experiment with wild-type and nonphosphobinding mutants of 14-3-3γ also suggest that 14-3-3 proteins bind endogenous nuclear O-GlcNAc moieties in human cells (SI Appendix, Fig. S11).

Characterization of the major O-GlcNAcylated binding partners of 14-3-3 will likely require extensive proteomic and cell biological efforts. As a first step toward this goal, we cloned wild-type and R57E mutant 14-3-3γ with tandem HA and Halo tags and performed GlcNDAz crosslinking in 293T cells. Consistent with our prior results (Fig. 3 C and D), wild-type HA-Halo-14-3-3γ crosslinked in a GlcNDAz-specific fashion (SI Appendix, Fig. S12). Importantly, R57E mutant HA-Halo-14-3-3γ exhibited GlcNDAz crosslinking nearly identical to that of wild-type protein (SI Appendix, Fig. S12), further reinforcing the conclusion that 14-3-3γ binds endogenous OGT substrates in a phosphorylation-independent manner. In future work, we anticipate that HA-Halo-14-3-3 constructs will allow stringent affinity purification and proteomic analyses of GlcNDAz-crosslinked proteins, revealing the major endogenous O-GlcNAcylated ligands of 14-3-3.

In summary, we report the systematic identification of candidate reader proteins that bind O-GlcNAc directly and specifically. In addition, our structures of 14-3-3/glycopeptide complexes provide a biophysical characterization of an O-GlcNAc-mediated interaction, laying the groundwork for future functional studies. The biochemical and structural characterization of reader proteins of other PTMs has greatly advanced our understanding of cell signaling (17, 18, 71–73). We expect that our results will similarly facilitate new analyses of 14-3-3 proteins and other candidate O-GlcNAc readers in a wide range of biological contexts.

Materials and Methods

Full proteomics datasets are available in Datasets S1–S5. X-ray crystal structures have been deposited in the Protein Data Bank under ID codes 6BYJ, 6BYK, 6BZD and 6BYL.

Additional details are available in the SI Appendix, Supporting Materials and Methods.