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Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase

Nature Chemical Biology volume 19, pages 1423–1431 (2023)Cite this article

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Abstract

The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein–protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain.

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Fig. 1: ProP-PD screening identified a specific OGT binding motif.
Fig. 2: OGT crystal structures reveal a binding site in the Int-D.
Fig. 3: Int-D binding site is a regulator of protein association and O-GlcNAcylation.
Fig. 4: Bioinformatic analysis of PxYx[I/L] motif-containing proteins.
Fig. 5: OGT Int-D binding site is a regulator of O-GlcNAc dynamics during nutrient deprivation and lactate production.

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Data availability

Atomic coordinates of the OGT4.5 co-crystal structures have been deposited in the RCSB Protein Data Bank under accession codes 8FE6 (OGT4.5:UDP-GlcNAc:CP37), 8FE7 (OGT4.5:UDP-GlcNAc:SMG9) and 8FUF (OGT4.5:UDP-GlcNAc:ZNF831). The data that support the findings of this study are available within the main text and its Supplementary Information file. Data are also available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We would like to thank members of the Jiang laboratory for insightful discussions and suggestions. This research was funded by National Institutes of Health (NIH) grant R01 GM121718 (J.J.), the University of Wisconsin-Madison Vilas Faculty Early Career Investigator Award (J.J.) and the National Science Foundation Graduate Research Fellowship under grant DGE-1747503 (C.M.B.). We also thank K. Satyshur for helpful suggestions on structure refinement; staff members of the Life Sciences Collaborative Access Team for assisting with X-ray data collection; and the University of Wisconsin Carbone Cancer Center Small Molecule Screening Facility and Drug Development Core Medicinal Chemistry Center (supported by NIH P30 CA014520) for use of the facilities and services. The authors acknowledge support from the National Genomics Infrastructure in Stockholm, funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and the SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. We thank N. E. Davey and L. Simonetti for support with the computational pipeline for next-generation sequencing data analysis.

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  1. These authors contributed equally: Connor M. Blankenship, Jinshan Xie.

Authors and Affiliations

  1. Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA

    Connor M. Blankenship, Jinshan Xie, Ao Wang & Jiaoyang Jiang

  2. Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden

    Caroline Benz & Ylva Ivarsson

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Contributions

C.M.B., J.X. and J.J. conceptualized the project. J.J. oversaw all aspects of experiments and manuscript preparation. C.M.B. and J.X. purified proteins and performed thermal shift assays. C.M.B. performed fluorescence polarization and bioinformatic analysis and carried out all cellular experiments. J.X. crystallized, structurally determined and computationally analyzed protein–peptide complexes and performed microscale thermophoresis analysis. C.B. performed phage display screening, next-generation sequencing and peptide motif analysis, with supervision by Y.I. A.W. measured the enzymatic activities of OGT variants. C.M.B., J.X. and J.J. wrote the manuscript. All authors participated in editing the manuscript.

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Correspondence to Jiaoyang Jiang.

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Extended data

Extended Data Fig. 1 OGT binding evaluation of motif-containing peptides.

(a) Sequences of one consensus (CP37) and seven natural motif-containing peptides along with their apparent binding affinities (EC50) from competitive fluorescence polarization assay using 5-FAM-SMG9 peptide and OGT4.5. Standard deviations from three biological replicates are shown. PxYx[I/L] motif in each peptide is highlighted in red. (b) Thermal shift assay showing the shift in denaturation temperature (Tm) of OGT and OGT4.5 with peptide incubation, n = 3. (c) Competitive fluorescence polarization assay between 5-FAM-SMG9 and motif-containing peptides AK9 (blue), DENND1B (green), OR2AG1 (purple), JMJD1C (brown), and CAMKMT (cyan) for OGT4.5 binding. Data are presented as individual measurements ± SD of mean of three biological replicates.

Source data

Extended Data Fig. 2

(a) Zoom-in view at the Int-D binding site demonstrating hydrophobic interactions between ZNF831 peptide (shown as sticks) and Int-D in OGT4.5 (shown as surface). Red and white colors represent the least and most hydrophobic areas, respectively. (b) Zoom-in view at the Int-D binding site demonstrating polar interactions between ZNF831 peptide (shown as purple sticks) and Int-D in OGT4.5 (shown as surface) with interacting OGT residues shown in sticks. 2Fo–Fc electron density map of ZNF831 peptide is shown as grey mesh and contoured at 1.0 σ, Fo–Fc electron density map is shown as green mesh and contoured at 3.0 σ. (c) Zoom-in view at the Int-D binding site demonstrating hydrophobic interactions between CP37 peptide (shown as sticks) and Int-D in OGT4.5 (shown as surface). Red and white colors represent the least and most hydrophobic areas, respectively. (d) Zoom-in view at the Int-D binding site demonstrating polar interactions between CP37 peptide (shown as blue sticks) and Int-D in OGT4.5 (shown as surface) with interacting OGT residues shown in sticks. 2Fo–Fc electron density map of CP37 peptide is shown as grey mesh and contoured at 1.0 σ, Fo–Fc electron density map is shown as green mesh and contoured at 3.0 σ. (e) Superimposition of OGT4.5:UDP-GlcNAc (4GZ5, grey), OGT4.5:UDP-GlcNAc:SMG9 (magenta), OGT4.5:UDP-GlcNAc:ZNF831 (purple), and OGT4.5:UDP-GlcNAc:CP37 (blue) crystal structures showing peptide binding to Int-D does not change the overall structure of OGT4.5. (f) A proposed model of a substrate peptide (shown as brown cartoon) binding to OGT4.5 (shown as surface, domains colored as in Fig. 1a). Int-D and TPR domain interactions facilitate substate glycosylation in the active site of OGT. G symbol represents GlcNAc moiety.

Extended Data Fig. 3 Evolutionary conservation analyses of OGT intervening domain.

(a) Sequences of 50 metazoan OGT homologs, including 25 homologs from vertebrates (highlighted in black box), were aligned by Clustal Omega58. Jalview59 was used to review the alignment results and generate the figure. Residues are colored according to the percentage identity in each column (>80 %, dark blue; > 60 %, blue; > 40%, light blue; < 40%, white). Int-D residues involved in motif interaction are highlighted in red boxes. (b) ConSurf60 analysis of metazoan (left) and vertebrate (right) OGT evolutionary conservation profile. Residues on the Int-D binding site are conserved among vertebrates but not invertebrates. Residues are colored from green to purple by variability across the aligned structures.

Extended Data Fig. 4 OGT Int-D mutants reduce peptide binding but retain intrinsic activity.

(a) Thermal shift assay showing the mutation induced change in OGT denaturation temperature (Tm). Data are presented as individual measurements ± SD of mean of three biological replicates. (b) Saturation fluorescence polarization (FP) binding assay of fluorescently labeled 5-FAM-SMG9 peptide with WT OGT (black) and mutants F723R (orange), F723E (blue), and I787R (green). Data are presented as mean values ± SD of three biological replicates. (c) Radiolabeled activity assay measuring integration of UDP-3H-GlcNAc onto CKII peptide by recombinantly purified WT OGT (WT) or OGT-N791A mutant (N791A). Data are presented as individual measurements ± SD of mean of three biological replicates.

Source data

Extended Data Fig. 5 The Int-D binding site is a motif-dependent regulator of protein association and O-GlcNAcylation.

Co-immunoprecipitation of cMyc-SMG9 with Flag-OGT (WT) or (a) Flag-OGT-I734R (I734R), (b) Flag-OGT-I787E (I787E), (c) Flag-OGT-I787E-N791A (2 M), from TRex-293 cells, followed by western blot. (d) Co-immunoprecipitation of endogenous OGA with Flag-OGT (W) or Flag-OGT-N791A (M) from TRex-293 cells, followed by western blot. (e) O-GlcNAcylation detection on CaMKMT from cells co-expressed with Flag-OGT (W) or Flag-OGT-N791A (M). HA-tagged CaMKMT was immunoprecipitated from TRex-293 cell lysate by anti-HA agarose and the O-GlcNAcylation was detected with the same GalT assay as SMG9. (f) O-GlcNAcylation detection on Flag-OGT (W), Flag-OGT-N791A (M), and Flag-HA-OGA-D175N. Flag-tagged OGT and OGA were immunoprecipitated from TRex-293 cell lysate by Flag-agarose, followed by western blot. All whole cell lysates have endogenous OGT knocked down. Blots are representative of at least three biological replicates.

Source data

Extended Data Fig. 6 Global O-GlcNAcylation is unperturbed by Int-D site mutation and single nutrient deprivation.

(a) Western blot detection of global O-GlcNAcylation in TRex-293 cells overexpressing OGT (WT), OGT-I787E (I787E), or OGT-I734R (I734R) with endogenous OGT knockdown (shOGT). Western blots of O-GlcNAcylation under high (H) and low (L) glucose (b) or serum (c) treatment with OGT (W) or OGT-N791A (M) overexpression. High nutrient conditions include DMEM with 4.5 g/L glucose and 10% FBS, low nutrient conditions include DMEM with 0.45 g/L glucose and 1% FBS. Blots are representative of at least three biological replicates.

Source data

Extended Data Fig. 7 Delayed response to nutrient deprivation in HeLa cells.

Western blot showing time course study of O-GlcNAcylation response to nutrient deprivation in HeLa cells stably expressing OGT (W) and OGT-N791A (M). Cells were induced for 48 hours prior to high or low nutrient treatment, then samples were collected at 3, 8, and 24 hours of treatment. Western blots are representative of three biological replicates.

Source data

Supplementary information

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Blankenship, C.M., Xie, J., Benz, C. et al. Motif-dependent binding on the intervening domain regulates O-GlcNAc transferase. Nat Chem Biol 19, 1423–1431 (2023). https://doi.org/10.1038/s41589-023-01422-2

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