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

Clifford A. Tolemana,1, Maria A. Schumachera,1, Seok-Ho Yub, Wenjie Zenga, Nathan J. Coxa, Timothy J. Smitha, Erik J. Soderblomc, Amberlyn M. Wandsb, Jennifer J. Kohlerb, and Michael Boycea,2

aDepartment of , Duke University School of Medicine, Durham, NC 27710; bDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390; and cDuke and Metabolomics Core Facility, Center for Genomic and Computational , Duke University, Durham, NC 27710

Edited by Carolyn R. Bertozzi, Stanford University, Stanford, CA, and approved April 23, 2018 (received for review December 24, 2017)

O-GlcNAc is an intracellular posttranslational modification that gov- Results erns myriad cell biological processes and is dysregulated in We developed a biochemical approach to test the hypothesis that diseases. Despite this broad pathophysiological significance, the O-GlcNAc is specifically recognized by mammalian reader pro- biochemical effects of most O-GlcNAcylation events remain unchar- teins. First, we derived a consensus O-GlcNAcylated acterized. One prevalent hypothesis is that O-GlcNAc moieties may sequence by aligning 802 mapped Ser-O-GlcNAc sites (34–36) be recognized by “reader” proteins to effect downstream signaling. (Fig. 1A)(www.phosphosite.org). We noted that a Pro-Val-Ser However, no general O-GlcNAc readers have been identified, leav- tripeptide observed previously in smaller datasets (37, 38) also ing a considerable gap in the field. To elucidate O-GlcNAc signaling emerged in our sequence, suggesting that this motif may be mechanisms, we devised a biochemical screen for candidate O-GlcNAc important for O-GlcNAc modification and/or recognition. We reader proteins. We identified several human proteins, including 14-3- reasoned that this consensus peptide, when glycosylated, might 3 isoforms, that bind O-GlcNAc directly and selectively. We demon- serve as biochemical “bait” for diverse O-GlcNAc reader pro- strate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and teins. Human OGT efficiently glycosylated a bait peptide con- we present the structures of 14-3-3β/α and γ bound to glycopeptides, taining the consensus sequence and a polyethylene glycol-biotin providing biophysical insights into O-GlcNAc-mediated –protein anchor (SI Appendix, Fig. S1A) with a single O-GlcNAc on the

interactions. Because 14-3-3 proteins also bind to phospho- and expected serine, as determined by (MS; Fig. 1B BIOCHEMISTRY phospho-, they may integrate information from O-GlcNAc and SI Appendix, Fig. S1B). The GlcNAc-binding lectin wheat and O- signaling pathways to regulate numerous germ agglutinin was affinity-enriched via pulldown with the bait physiological functions. glycopeptide, compared with the unglycosylated control peptide, even in the presence of excess nonspecific protein (Fig. 1C). These O-GlcNAc | reader proteins | 14-3-3 | enolase | EBP1 results indicated that our approach could selectively capture GlcNAc- binding proteins. -linked β-N-acetylglucosamine (O-GlcNAc) is an abundant Using the bait glycopeptide and MS proteomics, we affinity- posttranslational modification (PTM) of and threo- captured and identified dozens of endogenous nuclear and cy- O toplasmic proteins that selectively bound the O-GlcNAcylated nines on nuclear, cytoplasmic, and mitochondrial proteins, gov- erning diverse biological processes (1–3). In mammals, O-GlcNAc Significance is added by O-GlcNAc transferase (OGT) and removed by O- GlcNAcase (OGA), and is essential, as genetic ablation of ei- O-GlcNAc is an abundant, reversible posttranslational modification ther is lethal in mice (4–6). Aberrant O-GlcNAc cycling is (PTM) of nuclear and cytoplasmic proteins in animals and plants. also implicated in numerous human diseases, including (1, O-GlcNAc regulates a wide range of biological processes, and ab- – – – 7 9), (10 12), and neurodegeneration (13 16). errant O-GlcNAcylation is implicated in numerous human diseases. Despite this broad pathophysiological significance, the molec- However, key aspects of O-GlcNAc signaling remain poorly un- ular mechanisms of O-GlcNAc signaling are poorly understood. derstood. For example, it is not known whether “reader” proteins One prevailing hypothesis is that O-GlcNAc moieties, similar to exist to recognize and bind to O-GlcNAc, as is true for many other other intracellular PTMs, may be recognized by “reader” proteins PTMs. We used a biochemical method to identify candidate human that specifically bind O-GlcNAc to effect downstream functions O-GlcNAc reader proteins, and then characterized them at the (17, 18). However, although some examples of O-GlcNAc-mediated biochemical and biophysical levels. Our results address a signif- protein–protein interactions are known (19–26), no general icant gap in the field by revealing the biochemical O-GlcNAc readers have been identified, and no structure of any and structural basis for the recognition of O-GlcNAc by con- O-GlcNAc-mediated protein–protein interaction has been reported, served human proteins. leaving the biochemical and biophysical basis of O-GlcNAc recog- nition unclear. To address this knowledge gap, we devised a screen Author contributions: C.A.T., M.A.S., S.-H.Y., J.J.K., and M.B. designed research; C.A.T., M.A.S., S.-H.Y., N.J.C., T.J.S., E.J.S., and A.M.W. performed research; C.A.T., W.Z., and to discover candidate mammalian O-GlcNAc reader proteins. We A.M.W. contributed new reagents/analytic tools; C.A.T., M.A.S., S.-H.Y., N.J.C., E.J.S., discovered that multiple human proteins, including 14-3-3 iso- J.J.K., and M.B. analyzed data; and C.A.T. and M.B. wrote the paper. forms (17, 27, 28), bind selectively to O-GlcNAcylated substrates, The authors declare no conflict of interest. and we determined structures of human 14-3-3β/α and γ bound This article is a PNAS Direct Submission. to glycopeptides, providing atomic resolution information on an Published under the PNAS license. – O-GlcNAc-mediated protein protein interaction. Remarkably, Data deposition: The atomic coordinates and structure factors have been deposited in the the 14-3-3 O-GlcNAc-binding pocket overlaps with its well-known , www.wwpdb.org (PDB ID codes 6BYJ, 6BYK, 6BZD, and 6BYL). phosphorylated ligand binding site (17, 27, 28). Therefore, 14-3- 1C.A.T. and M.A.S. contributed equally to this work. 3 proteins may serve as bifunctional readers, integrating in- 2To whom correspondence should be addressed. Email: [email protected]. formation in the previously documented, extensive crosstalk This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. between O-GlcNAcylation and O- pathways (1, 1073/pnas.1722437115/-/DCSupplemental. 29–33).

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C O-GlcNAc D O-GlcNAc

O-GlcNAc E F nuclear cytoplasmic O-GlcNAc silver stain importin-5

importin- 1

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 +1Mg2 . See also SI Appendix, Fig. S1B.(C) Fluorescein-tagged wheat germ agglutinin was incubated with glycopeptide or mock-modified peptide (±O-GlcNAc), without (Left)orwith(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 glyco- peptide 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.

probe in extracts from three different human cell lines (Fig. 1 D EBP1, and 14-3-3 share evolutionarily conserved O-GlcNAc- and E and SI Appendix, Fig. S1C). Then, we inspected our MS binding properties. datasets for the proteins most enriched by the O-GlcNAcylated To determine whether α-enolase, EBP1, and 14-3-3 bind O- bait in at least two cell types (SI Appendix, Fig. S1C). One protein GlcNAc moieties in vivo, we created expression constructs encod- β meeting these criteria was importin- 1, which mediates nuclear ing mCherry fused to the Ser-O-GlcNAc bait sequence and a cargo trafficking (39) and was previously shown to interact di- rectly with O-GlcNAc moieties on nuclear pore proteins in hu- man cells (40). We confirmed by immunoblot (IB) that importin- β1 and its homolog importin-5 were specifically enriched by the A B bait glycopeptide (Fig. 1F). Interestingly, several other proteins O-GlcNAc - enriched in our experiments exist as dimers or higher-order olig- 14-3-3 14-3-3 / omers, possibly indicating that avidity effects from the densely glycoprotein-decorated beads contributed to their successful O- GlcNAc-specific purification (SI Appendix,Fig.S1C). These re- sults 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 and proliferation induced by the re- PP1 (MBP) C glycopeptide ceptor ErbB3 (42), and 14-3-3 proteins bind

phosphoserine/threonine moieties to govern numerous processes, Binding affinities for selected including growth factor signaling, mitotic exit, and apoptosis (17, D protein/glycopeptide interactions

Protein KD 27, 28). We verified that the bait glycopeptide specifically enriched mP control A B -enolase 183 ± 54 both endogenous (Fig. 2 ) and recombinant-purified (Fig. 2 ) EBP1 172 ± 8 forms of human α-enolase, EBP1, and 14-3-3β/α and γ (repre- 14-3-3 324 ± 12 sentative paralogs), demonstrating that they bind O-GlcNAc di- rectly, and not through bridging proteins. We confirmed this conclusion using fluorescence anisotropy (FA), which revealed [14-3-3 saturable binding of all three proteins to the glycopeptide, but not to the unglycosylated peptide (Fig. 2 C and D and SI Appendix, Fig. 2. Human protein binds O-GlcNAc directly and selectively. (A) Glyco- peptide pulldowns from 293T extracts were analyzed by IB. Tubulin is a Fig. S2). Extending these observations, we found that human α negative control. (B) Recombinant-purified proteins were analyzed by gly- -enolase, EBP1, and 14-3-3 also directly bound a consensus copeptide pulldown and IB. Protein -1 (PP1) (not enriched in any glycopeptide derived from 676 mapped threonine-O-GlcNAc sites MS sample) is a negative control. (C) Fluorescein-labeled bait peptide was (34–36) (SI Appendix,Fig.S3)(www.phosphosite.org), and that mock- (blue) or O-GlcNAc-modified (red), and binding to 14-3-3γ was ana- the murine orthologs of all three proteins were also specifically lyzed by FA. Representative traces from triplicate experiment are shown. mP, affinity-enriched by bait glycopeptides (SI Appendix, Fig. S4). millipolarization. (D) Dissociation constants for glycopeptide interactions Together, these results demonstrate that mammalian α-enolase, with α-enolase, EBP1, and 14-3-3γ, determined by FA.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1722437115 Toleman et al. Downloaded by guest on October 2, 2021 A B 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, e, 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 C (SI Appendix, Fig. S5B). IBs confirmed the GlcNDAz-specific D 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 pro- vided 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 ques- tion, 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 glyco- Fig. 3. Candidate reader proteins bind O-GlcNAcylated substrates in human peptide pulldowns specifically enriched all family members, ex- cells. (A) 293T cells were transfected with mCherry, mCherry-Ser-bait-myc, cept σ, from mammalian cell extracts (Figs. 2A and 4A and SI or mCherry-Ala-bait-myc. Lysates were analyzed by anti-myc IP and IB. Appendix, Fig. S1C). Moreover, glycopeptide pulldowns (Fig. 4B) (B) 293T cells were transfected as in A, incubated 24 h, and treated with vehicle, C SI Appendix μ μ and FA (Fig. 4 and , Fig. S6) with recombinant- 50 M 5SGlcNAc (OGT inhibitor), or 50 M Thiamet-G (OGA inhibitor) for 6 h. purified protein confirmed the direct and selective binding of 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 two additional 14-3-3 isoforms to O-GlcNAc, with no saturable binding to the unglycosylated peptide detected. The affinities of BIOCHEMISTRY from the corresponding input sample (noted directly beneath each IB). D SI Ap- (C) AGX1(F383G)-expressing HeLa cells (40) were treated with GlcNDAz 14-3-3 isoforms for the bait glycopeptide (Fig. 2 and precursor and ultraviolet (or not, negative control). Lysates were analyzed by pendix, Fig. S6), although moderate, are comparable to those anti-O-GlcNAc IP, SDS/PAGE, and silver stain. Gel slices of defined MW ranges reported previously for some phosphopeptide ligands from were analyzed by spectral index quantitation MS proteomics (74). Graph physiological binding partners (51, 52). To further test the po- depicts selected proteins identified in the 100–200-kDa gel region and tential physiological relevance of the affinities we observed for enriched more than fivefold in the +UV sample versus the −UV (control) 14-3-3 and model glycopeptides, we synthesized previously vali- sample. Y-axis indicates protein abundance, as quantified by mean ion dated 14-3-3-binding phosphopeptides from the current (MIC). X-axis indicates the ratio (fold-enrichment) of each protein transmembrane conductance regulator and the in the +UV/−UV samples. Proteins identified in the +UV sample but absent factor Snail (51, 52) (SI Appendix, Fig. S1A) and examined them − ∞ from the UV control are denoted as enrichment. Axis breaks indicate scale under identical FA assay conditions. Consistent with prior re- changes only. See also SI Appendix,Fig.S5.(D) AGX1(F383G)-expressing ports (51, 52), we observed weak binding with both cystic fibrosis 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, transmembrane conductance regulator and Snail ligands, with affinities lower than those we observed for the model glyco- uncrosslinked 14-3-3. Arrows, 14-3-3 crosslinks (i.e., O-GlcNAc-mediated SI Appendix interactions). ( , Fig. S7), demonstrating that the 14-3-3/ glycopeptide affinities we observe are on par with some physio- logical 14-3-3/phosphopeptide interactions. By analogy to prior myc . Endogenous 14-3-3, α-enolase, and EBP1 coim- studies comparing phosphopeptides and full-length phospho- munoprecipitated (IP-ed) with transfected mCherry-bait, but proteins (53), we expect that 14-3-3 isoforms likely bind signifi- not with a serine→ “mutant” bait (Fig. 3A). Moreover, a cantly tighter to native, full-length glycosylated partner proteins specific small molecule inhibitor of OGT (5SGlcNAc) (43) than they do to our consensus glycopeptides, which were designed greatly reduced the interaction between candidate reader pro- to capture a variety of reader proteins, and hence were not se- teins and mCherry-bait, whereas an OGA inhibitor (Thiamet- lected or optimized for 14-3-3 binding in particular (Fig. 1A and SI G) (44) had little effect (Fig. 3B). These results indicate that Appendix,Fig.S3A). constitutive O-GlcNAcylation of the bait peptide sequence is Next, to elucidate the biophysical basis of O-GlcNAc binding efficient, and that endogenous α-enolase, EBP1, and 14-3-3 can by 14-3-3 paralogs, we obtained X-ray crystal structures of serine bind O-GlcNAc moieties in human cells. and threonine bait glycopeptides bound to 14-3-3β/α or γ (Fig. 4 To determine whether candidate reader proteins interact D and E and SI Appendix, Figs. S8–10). Strikingly, the glyco- with native OGT substrates in living cells, we used a chemical peptides occupy the same amphipathic groove of 14-3-3, where biology strategy (40) to covalently capture endogenous O- phosphorylated ligands bind (54). In addition, to protein/peptide GlcNAc-mediated protein–protein interactions. Briefly, in this backbone contacts, 14-3-3 makes 10 hydrogen bonds with the approach, human cells are metabolically labeled with a dia- glycan (Fig. 4 D and E), revealing the biophysical basis for O- zirine-bearing GlcNAc analog (GlcNDAz), which they convert GlcNAc-selective binding (Figs. 1–3). Moreover, the glyco- to a nucleotide- species used by OGT to modify its nat- peptide and protein conformations were nearly identical in ural substrates (40). Short UV treatment of GlcNDAz-labeled the 14-3-3β/α and γ cocomplexes (Fig. 4 D, E,andI and SI cells covalently crosslinks proteins within ∼2–4ÅoftheO- Appendix,Fig.S9), providing a structural explanation for the GlcNDAz moiety, capturing direct binding partners but not conserved O-GlcNAc binding property of 14-3-3 isoforms (Fig. nonspecific proteins (25, 26, 40). We performed GlcNDAz 4A). Remarkably, the bound O-GlcNAc moiety is essentially su- crosslinking on human cells, IP-ed lysates with an anti-O-GlcNAc perimposable on the O-phosphate group of prior phosphopeptide- , separated IP-ed proteins by SDS/PAGE, and used bound structures of 14-3-3, with similar residues contacting both quantitative proteomics to identify proteins within gel slices of PTMs, despite their considerable steric and electronic differences defined molecular weight (MW) ranges (SI Appendix,Fig.S5). (Fig. 4F).

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DE

FG

HI

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)orFA(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)or2.5σ (14-3-3β/α)(Right). (F) Overlay of 14-3-3γ-glycopeptide (from D) and 14-3-3γ- phosphopeptide from (magenta protein, red phosphopeptide; PDB 4J6S) (75). O-GlcNAc and O-phosphate moieties are highlightedby 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.

We next used our structures to design mutations that uncouple glycopeptide (Fig. 4 D and E). Indeed, both N178Y and V181W the O-GlcNAc and O-phosphate binding properties of 14-3-3. 14-3-3γ failed to bind O-GlcNAcylated ligands in a pulldown Mutating R57, R132, or Y133 (14-3-3γ numbering) to glutamate assay (Fig. 4G). Together, these results provide biochemical and is known to abolish phospho-ligand binding because of electro- structural insights into selective, O-GlcNAc-mediated protein– static repulsion (55, 56). We reasoned that these mutants might protein interactions, and reveal the biophysical basis of glycan still bind uncharged O-GlcNAc moieties. Indeed, the R57E, binding by 14-3-3 proteins, a property distinct and separable from R132E, and Y133E 14-3-3γ mutants selectively bound glyco- O-phosphate binding. peptides in pulldown and FA experiments (Fig. 4 G and H and SI Finally, we leveraged our structural and mutagenesis data to Appendix, Fig. S6) comparably to wild type (Fig. 2). Further- further investigate the relationship between 14-3-3 and O- more, we obtained the structure of a 14-3-3γ R57E-glycopeptide GlcNAc signaling in human cells. Consistent with an earlier re- complex, which confirmed that O-GlcNAc binding is preserved port (57), we found that expressed wild-type 14-3-3γ was broadly without significant conformational changes (Fig. 4I). In a re- distributed throughout the nucleus and cytoplasm, whereas the ciprocal experiment, we created N178Y and V181W mutants, R57E and Y133E 14-3-3γ mutants, which bind O-GlcNAc (Fig. 4 which we predicted would not bind O-GlcNAc, hypothesizing G–I and SI Appendix,Fig.S6) but not O-phosphate (55, 56), localize that the bulkier tyrosine or residue would steri- specifically to the nucleus (SI Appendix,Fig.S11). Moreover, both cally disrupt the key contacts made by N178 and V181 to the mutants were dispersed throughout the cell on treatment with either

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1722437115 Toleman et al. Downloaded by guest on October 2, 2021 of two structurally unrelated small molecule OGT inhibitors (SI ligands. Moreover, because 14-3-3 isoforms homo- and hetero- Appendix,Fig.S11), suggesting that their localization depends on dimerize (17, 27, 28), the two amphipathic grooves of a single endogenous glycosylated (but not phosphorylated) nuclear ligands. 14-3-3 dimer could conceivably bind one O-GlcNAc and one O- These data further support a role for 14-3-3 as O-GlcNAc reader phosphate, respectively, when interacting with the many proteins proteins in live human cells. known to be both phosphorylated and glycosylated (1, 29–33). Indeed, some native binding partners of 14-3-3 are phosphory- Discussion lated at multiple sites, providing both high- and lower-affinity Despite their importance, the biochemical mechanisms of O- phospholigands for binding 14-3-3 dimers, permitting combina- GlcNAc signaling remain incompletely understood. To address torial tuning of cell signaling through multiple PTMs on a single this knowledge gap, we tested the hypothesis that the O-GlcNAc protein (52, 66, 67). We speculate that O-GlcNAc moieties may modification is specifically recognized by mammalian reader similarly serve as high- or lower-affinity 14-3-3 binding sites, proteins. Several pioneering studies from the Lefebvre group perhaps in combination with phosphorylation sites, on endoge- previously reported that Hsp70 family chaperones bind GlcNAc- nous human proteins. Testing these hypotheses will be an im- agarose resin and associate with O-GlcNAcylated mammalian portant goal for future studies. proteins in a nutrient- and stress-responsive manner (58–62). The natural O-GlcNAcylated binding partners of 14-3-3 pro- However, whether Hsp70 chaperones or other human proteins teins remain to be identified. However, our proteomics results serve as direct and specific O-GlcNAc-binding readers remained revealed several known endogenous nuclear glycoproteins in the unclear. same GlcNDAz-crosslinked, high-molecular-weight complexes Our results demonstrate that diverse human proteins bind O- as endogenous 14-3-3 (Fig. 3C and SI Appendix, Fig. S5). Im- GlcNAcylated substrates directly and selectively. For example, portantly, the chemical mechanism and short crosslinking radius α-enolase binds O-GlcNAcylated substrates (Figs. 1–3) and is of the GlcNDAz reagent ensure that these in vivo interactions itself O-GlcNAcylated (8), indicating that may are mediated by direct binding to glycans (40). These data sug- govern through regulated protein–protein interactions. gest that 14-3-3 may exploit high O-GlcNAc ligand avidity to In support of this possibility, our glycopeptide pulldowns enriched bind directly to heavily glycosylated nucleoporins or other nuclear several glycolytic and pentose phosphate pathway (SI OGT substrates (68–70). Consistent with this hypothesis, our Appendix,Fig.S1C), consistent with the well-documented role microscopy experiment with wild-type and nonphosphobinding γ of O-GlcNAc signaling in nutrient sensing (1, 2). Similarly, mutants of 14-3-3 also suggest that 14-3-3 proteins bind en- SI Ap- EBP1 binds O-GlcNAcylated ligands (Figs. 1–3) and interacts dogenous nuclear O-GlcNAc moieties in human cells ( pendix,Fig.S11). with known OGT substrates, including the transcriptional regulators BIOCHEMISTRY E2F1 and Sin3A (63–65), suggesting that O-GlcNAcylation may Characterization of the major O-GlcNAcylated binding part- influence ErbB3 signaling through regulated multiprotein complexes ners of 14-3-3 will likely require extensive proteomic and cell of EBP1. The structural basis and functional consequences of biological efforts. As a first step toward this goal, we cloned wild- γ O-GlcNAc binding by α-enolase and EBP1 are currently under type and R57E mutant 14-3-3 with tandem HA and Halo tags investigation. and performed GlcNDAz crosslinking in 293T cells. Consistent C D The 14-3-3 isoforms, also identified in our screen as O- with our prior results (Fig. 3 and ), wild-type HA-Halo-14-3- 3γ crosslinked in a GlcNDAz-specific fashion (SI Appendix, Fig. GlcNAc binders, play central roles in many physiological pro- γ cesses, including mitogenic signaling, progression, and S12). Importantly, R57E mutant HA-Halo-14-3-3 exhibited cell death (17, 27, 28). Our biophysical studies with 14-3-3 pro- GlcNDAz crosslinking nearly identical to that of wild-type pro- – tein (SI Appendix, Fig. S12), further reinforcing the conclusion vide structures of O-GlcNAc-mediated protein protein in- γ teraction, lending insight into the signaling mechanisms of 14-3-3 that 14-3-3 binds endogenous OGT substrates in a phosphory- and O-GlcNAc alike. On the basis of these results, we suggest lation-independent manner. In future work, we anticipate that that 14-3-3 proteins are well-suited to serve as general O- HA-Halo-14-3-3 constructs will allow stringent affinity purifica- GlcNAc readers. Our structures reveal that the amphipathic tion and proteomic analyses of GlcNDAz-crosslinked proteins, groove of 14-3-3 is especially well equipped to bind the Pro-Val- revealing the major endogenous O-GlcNAcylated ligands of Ser/Thr motif (Fig. 1A and SI Appendix, Fig. S3) present in 14-3-3. hundreds of natural OGT substrates (37, 38). Specifically, the In summary, we report the systematic identification of candi- in this glycopeptide motif occupies a shallow hydropho- date reader proteins that bind O-GlcNAc directly and specifically. bic pocket of 14-3-3 that likely would not tolerate large or polar In addition, our structures of 14-3-3/glycopeptide complexes pro- side chains (Fig. 4 and SI Appendix, Fig. S9). Similarly, the valine, vide a biophysical characterization of an O-GlcNAc-mediated although surface exposed, lies near the conserved L225 and the interaction, laying the groundwork for future functional studies. Cβ atoms of D228 and N229 (14-3-3γ numbering), making sev- The biochemical and structural characterization of reader proteins SI Appendix of other PTMs has greatly advanced our understanding of cell eral favorable contacts (Fig. 4 and , Fig. S9). Im- – portantly, specific 14-3-3 residues, (e.g., D129, N178, and E185, signaling (17, 18, 71 73). We expect that our results will similarly 14-3-3γ numbering) participate in O-GlcNAc binding but not O- facilitate new analyses of 14-3-3 proteins and other candidate phosphate binding (Fig. 4 and SI Appendix, Fig. S9), consistent O-GlcNAc readers in a wide range of biological contexts. with our observation that the O-GlcNAc and O-phosphate binding properties of 14-3-3 isoforms are genetically separable Materials and Methods G–I SI Appendix (Fig. 4 and , Fig. S9) (55, 56). Our results also Full proteomics datasets are available in Datasets S1–S5. X-ray crystal struc- demonstrate that multiple 14-3-3 isoforms from different mam- tures have been deposited in the Protein Data Bank under ID codes 6BYJ, malian species bind Ser- and Thr-O-GlcNAc moieties specifically – SI Appendix – 6BYK, 6BZD and 6BYL. and directly (Figs. 2 4 and , Figs. S2 S4), likely Additional details are available in the SI Appendix, Supporting Materials through nearly identical biophysical contacts (Fig. 4I and SI and Methods. Appendix, Fig. S9). Therefore, the amphipathic groove of mam- malian 14-3-3 isoforms is an evolutionarily conserved O-GlcNAc- ACKNOWLEDGMENTS. We thank Suzanne Walker (Harvard Medical School) binding protein module, in addition to its well-established and Pei Zhou (Duke University School of Medicine) for chemicals and plasmids, O-phosphate-binding role. Benjamin Swarts (Central Michigan University) for Ac45SGlcNAc, the Ad- O-GlcNAc and O-phosphate often compete for identical or vanced Light Source beamline 8.3.1 for data collection, and James Alvarez, nearby residues on numerous substrates, producing a complex Jen-Tsan Ashley Chi, Benjamin Swarts, and members of the M.B. laboratory – for helpful suggestions. This work was supported by a Rita Allen Founda- functional interplay between these PTMs (1, 29 33). Our results tion Scholar Award and NIH Grants 1R01GM118847-01 and UL1TR001117 raise the possibility that 14-3-3 isoforms act as bifunctional (to M.B.), and grants from the NIH (R21DK112733) and Welch Foundation reader proteins of specific O-GlcNAcylated or phosphorylated (I-1686) (to J.J.K.).

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