Biology subjects

Alteen, M. G.

Publications and source records attributed to Alteen, M. G..

3 recordsLinked to original sources

bioRxiv2023

Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is therefore regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of [~]108 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a non-competitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest an unprecedented mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity. SIGNIFICANCE STATEMENTThousands of proteins within humans are modified by the monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAc regulates cellular physiology and is being pursued to create therapeutics. Remarkably, only one enzyme, O-GlcNAc transferase (OGT), installs O-GlcNAc and its regulation is poorly understood. By affinity selection using a vast peptide library, we uncover an amino acid sequence motif that drives binding of polypeptides to OGT. An OGT-peptide complex shows how this motif binds to an allosteric site proximal to the active site and inhibits OGT in an unprecedented manner. Given the distribution of this sequence motif within the human proteome proteins containing this motif likely regulate the activity of OGT, outlining a new mode by which OGT is controlled and opening new avenues for research.

biochemistry↗

bioRxiv2023

Conformationally Adaptive Dyes Enable Protein-Adaptive Differential Scanning Fluorometry

Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding. One-Sentence SummaryNext generation protein-adaptive DSF (paDSF) enables rapid and general measurements of protein stability and dynamics.

biochemistry↗

bioRxiv2022

Expanding the Huntingtons disease research toolbox; validated huntingtin subdomain constructs for biochemical and structural investigation of the huntingtin protein

Huntingtons disease is characterised by CAG expansion in the huntingtin gene above a critical threshold of ~35 repeats, resulting in polyglutamine expansion of the huntingtin protein (HTT). The biological role of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part, due to challenges characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of rationally designed, high-quality recombinant HTT subdomains; one spanning the N-terminal HEAT and bridge domains (NTD) and the second spanning the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with 40 kDa HTT-associated protein (HAP40). We report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD-HAP40 complex binds the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validate the interaction between the CTD and HAP40 using a luciferase two-hybrid assay and use subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will enable the wider HD community to study fundamental HTT biology, discover new macromolecular or small-molecule binding partners and map interaction sites across this very large protein.

biochemistry↗