Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity

Forrest A Hammel ¹, N Connor Payne ^2,3, Victoria M Marando ¹

⁰Department of Microbiology, Harvard Medical School, Boston, Massachusetts 02115, United States.

Journal of the American Chemical Society +

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September 14, 2024

View abstract on PubMed

Summary

O-GlcNAc transferase (OGT) binds host cell factor 1 (HCF-1) with picomolar affinity. This interaction enables potent inhibition of OGT activity, leading to a novel, reversible inhibitor for cellular studies.

Area Of Science

Biochemistry
Molecular Biology
Enzymology

Background

O-GlcNAc transferase (OGT) is a crucial enzyme regulating cellular processes through protein glycosylation.
Host cell factor 1 (HCF-1) is a known OGT substrate and interactor, but its binding affinity to OGT remains unquantified.
Understanding OGT-HCF-1 interactions is key to deciphering OGT's regulatory mechanisms.

Purpose Of The Study

To quantify the binding affinity between OGT and its prominent binding partner, HCF-1.
To elucidate the molecular basis of the OGT-HCF-1 interaction.
To develop a novel OGT inhibitor based on the HCF-1 binding motif.

Main Methods

Time-resolved Förster resonance energy transfer (TR-FRET) assay to measure OGT-ligand binding kinetics.
Site-directed mutagenesis of OGT's tetratricopeptide repeat (TPR) domain to identify key binding residues.
In vitro and cellular assays to assess OGT inhibition by HCF-1 derived peptides.
Development of a genetically encoded, inducible OGT inhibitor.

Main Results

An HCF-1 derived polypeptide (HCF3R) exhibits picomolar binding affinity (K_D ≤ 85 pM) to OGT.
Conserved asparagines within OGT's TPR domain are critical for high-affinity HCF-1 binding, with mutations reducing affinity by over 5 orders of magnitude.
HCF3R potently inhibits OGT activity both in vitro and in cell-based assays.
A novel, genetically encoded, inducible, and reversible OGT inhibitor was successfully developed.

Conclusions

The OGT-HCF-1 interaction is characterized by exceptionally high affinity, driven by specific molecular contacts.
This high-affinity interaction can be leveraged to create potent OGT inhibitors.
The developed inducible inhibitor provides a valuable tool for studying OGT function in a tunable and reversible manner.