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Instruments / Technical Area	Publication Details	Published	Actions
I03-Macromolecular Crystallography	Cooperativity between the orthosteric and allosteric ligand binding sites of RORγt Rens M. J. M. De Vries , Femke A. Meijer , Richard G. Doveston , Iris A. Leijten-Van De Gevel , Luc Brunsveld Proceedings Of The National Academy Of Sciences 118 DOI: 10.1073/pnas.2021287118 Diamond Proposal Number(s): [19800] Show Abstract ▼ Abstract: Cooperative ligand binding is an important phenomenon in biological systems where ligand binding influences the binding of another ligand at an alternative site of the protein via an intramolecular network of interactions. The underlying mechanisms behind cooperative binding remain poorly understood, primarily due to the lack of structural data of these ternary complexes. Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs for RORγt, a nuclear receptor associated with the pathogenesis of autoimmune diseases. To provide the crucial structural insights, we solved 12 crystal structures of RORγt simultaneously bound to various orthosteric and allosteric ligands. The presence of the orthosteric ligand induces a clamping motion of the allosteric pocket via helices 4 to 5. Additional molecular dynamics simulations revealed the unusual mechanism behind this clamping motion, with Ala355 shifting between helix 4 and 5. The orthosteric RORγt agonists regulate the conformation of Ala355, thereby stabilizing the conformation of the allosteric pocket and cooperatively enhancing the affinity of the allosteric inverse agonists.	Feb 2021
I24-Microfocus Macromolecular Crystallography	Fluorescence anisotropy-based tethering for discovery of protein–protein interaction stabilizers Eline Sijbesma , Bente A. Somsen , Galen P. Miley , Iris A. Leijten-Van De Gevel , Luc Brunsveld , Michelle R. Arkin , Christian Ottmann Acs Chemical Biology DOI: 10.1021/acschembio.0c00646 Open Access Show Abstract ▼ Abstract: Protein–protein interaction (PPI) networks are fundamental for cellular processes. Small-molecule PPI enhancers have been shown to be powerful tools to fundamentally study PPIs and as starting points for potential new therapeutics. Yet, systematic approaches for their discovery are not widely available, and the design prerequisites of “molecular glues” are poorly understood. Covalent fragment-based screening can identify chemical starting points for these enhancers at specific sites in PPI interfaces. We recently reported a mass spectrometry-based disulfide-trapping (tethering) approach for a cysteine residue in the hub protein 14–3–3, an important regulator of phosphorylated client proteins. Here, we invert the strategy and report the development of a functional read-out for systematic identification of PPI enhancers based on fluorescence anisotropy (FA-tethering) with the reactive handle now on a client-derived peptide. Using the DNA-binding domain of the nuclear receptor Estrogen Related Receptor gamma (ERRγ), we target a native cysteine positioned at the 14–3–3 PPI interface and identify several fragments that form a disulfide bond to ERRγ and stabilize the complex up to 5-fold. Crystallography indicates that fragments bind in a pocket comprised of 14–3–3 and the ERRγ phosphopeptide. FA-tethering presents a streamlined methodology to discover molecular glues for protein complexes.	Nov 2020

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