Cooperativity between the orthosteric and allosteric ligand binding sites of RORγt Rens M. J. M. de Vriesa,b,1, Femke A. Meijera,b,1, Richard G. Dovestona,b,c,d, Iris A. Leijten-van de Gevela,b, and Luc Brunsvelda,b,2 aLaboratory of Chemical Biology, Department of Biomedical Engineering, Technische Universiteit Eindhoven, 5612 AZ Eindhoven, The Netherlands; bInstitute for Complex Molecular Systems, Technische Universiteit Eindhoven, 5612 AZ Eindhoven, The Netherlands; cLeicester Institute of Structural and Chemical Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; and dSchool of Chemistry, University of Leicester, LE1 7RH Leicester, United Kingdom Edited by Robert J. Fletterick, University of California, San Francisco, CA, and approved December 31, 2020 (received for review October 12, 2020) Cooperative ligand binding is an important phenomenon in bio- endogenous and synthetic small molecules (17) and constitute logical systems where ligand binding influences the binding of attractive drug targets, with 16% of all drugs targeting this pro- another ligand at an alternative site of the protein via an intra- tein class (18). Mechanistical understanding and exploiting of molecular network of interactions. The underlying mechanisms cooperative dual ligand binding in NRs harbors great potential behind cooperative binding remain poorly understood, primarily for drug development. An interesting NR in this context is the due to the lack of structural data of these ternary complexes. retinoic acid receptor-related orphan receptor γ t (RORγt) that Using time-resolved fluorescence resonance energy transfer (TR-FRET) studies, we show that cooperative ligand binding occurs plays an essential role in the differentiation of T helper 17 for RORγt, a nuclear receptor associated with the pathogenesis of (Th17) cells, associated with the pathogenesis of autoimmune – γ autoimmune diseases. To provide the crucial structural insights, diseases (19 21). Inhibition of ROR t with small molecules, in we solved 12 crystal structures of RORγt simultaneously bound order to disrupt the Th17/IL-17 pathway, is a promising strategy to various orthosteric and allosteric ligands. The presence of the toward reducing the inflammatory response (20, 22–30). The orthosteric ligand induces a clamping motion of the allosteric RORγt ligand binding domain (LBD) has been shown to contain pocket via helices 4 to 5. Additional molecular dynamics simula- both a clearly defined canonical, orthosteric binding site tions revealed the unusual mechanism behind this clamping mo- (Fig. 1 A and B), accessible for both endogenous and synthetic CHEMISTRY tion, with Ala355 shifting between helix 4 and 5. The orthosteric compounds (24, 31–33), and a high-affinity second binding site, γ ROR t agonists regulate the conformation of Ala355, thereby sta- termed allosteric pocket, formed by helices 3, 4, and 11 and bilizing the conformation of the allosteric pocket and coopera- reoriented helix 12 (H12), to which allosteric inverse agonists tively enhancing the affinity of the allosteric inverse agonists. can bind (Fig. 1C) (34–41). The crystal structures of the RORγt nuclear receptors | RORγt | allosteric modulators | structure elucidation | LBD bound to either an orthosteric or allosteric modulator drug discovery Significance BIOCHEMISTRY llosteric ligands bind to pockets on proteins that typically do Anot overlap with the canonical, orthosteric binding pockets RORγt is a nuclear receptor associated with several diseases. that are usually targeted by endogenous ligands (1, 2). Thus, Various synthetic ligands have been developed that target the allosteric ligands exert their effects via different structural modes canonical orthosteric or a second, allosteric pocket of RORγt. of action (1–3). This can convey advantages over orthosteric li- We show that orthosteric and allosteric ligands can simulta- γ gands in terms of potency, because competition with endogenous neously bind to ROR t and that their potency is positively ligands is removed, and selectivity, because allosteric sites are influenced by the other ligand, a phenomenon called cooper- less conserved across protein families (1). Molecules that ative dual ligand binding. The mechanism behind cooperative target allosteric pockets are therefore of high interest for drug binding in proteins is poorly understood, primarily due to the lack of structural data. We solved 12 crystal structures of development. Such ligands have been identified for several im- γ portant protein classes, like G protein coupled receptors ROR t, simultaneously bound to various orthosteric and allo- steric ligands. In combination with molecular dynamics, we (GPCRs) and kinases (4–6), with some of those compounds reveal a mechanism responsible for the cooperative binding developed into marketed drugs (7, 8). behavior. Our comprehensive structural studies provide unique Simultaneous binding of an endogenous, orthosteric ligand insights into how cooperative binding occurs in proteins. and an allosteric drug at different binding sites (dual ligand binding) is a fascinating pharmacological concept since this can Author contributions: R.M.J.M.d.V., F.A.M., and L.B. designed research; R.M.J.M.d.V., modulate the overall physiological effect of the drug. Of par- F.A.M., and I.A.L.-v.d.G. performed research; R.G.D. contributed new reagents/analytic ticular significance are cooperative dual ligand binding events tools; R.M.J.M.d.V., F.A.M., R.G.D., and L.B. analyzed data; and R.M.J.M.d.V., F.A.M., where binding of one ligand enhances that of the other (9–11), as R.G.D., and L.B. wrote the paper. observed for GPCR ligands in particular (12, 13). However, Competing interest statement: L.B. is scientific cofounder of Ambagon Therapeutics, a 14- 3-3 drug discovery company. F.A.M., R.G.D., and L.B. are coinventors of patent detailed structural insights into the mechanics of cooperative WO2020149740: Substituted heterocyclic compounds and their use as retinoid-related ligand binding remain scarce (14). This, in part, results from the orphan receptor (ROR) gamma-t inhibitors. absence of high-resolution structural data, required to visualize This article is a PNAS Direct Submission. the effects of dual ligand binding. Better structural understand- This open access article is distributed under Creative Commons Attribution-NonCommercial- ing of cooperativity in dual ligand binding is therefore required NoDerivatives License 4.0 (CC BY-NC-ND). to accelerate the development of new allosteric drugs. 1R.M.J.M.d.V. and F.A.M. contributed equally to this work. Dual ligand binding has occasionally been observed for nu- 2To whom correspondence may be addressed. Email: [email protected]. clear receptors (NRs), but there is no clear mechanistic under- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ standing of connected cooperative effects (15, 16). NRs are a doi:10.1073/pnas.2021287118/-/DCSupplemental. class of transcription factors that can be modulated by Published February 3, 2021. PNAS 2021 Vol. 118 No. 6 e2021287118 https://doi.org/10.1073/pnas.2021287118 | 1of9 Downloaded by guest on October 2, 2021 Fig. 1. (A) Conceptual representation of the RORγt LBD which is intrinsically active in the apo state, inducing cofactor binding. In the presence of an orthosteric agonist (blue), cofactor binding is further increased, while in the presence of an allosteric inverse agonist (pink), cofactor binding is blocked. In the presence of both an orthosteric and an allosteric ligand, the cofactor blockage is most efficient because of cooperative dual ligand binding. (B) Crystal structure of the RORγt LBD with the agonist 25-OH bound to the orthosteric site (blue sticks; PDB entry 3L0L). (C) Crystal structure of the RORγt LBD with inverse agonist MRL-871 bound to the allosteric site (red sticks; PDB entry 5C4O). The structures show that the orthosteric and allosteric binding sites do not overlap and highlight the prominently different orientation of H12. (D) Chemical structures of RORγt orthosteric agonists (20-OH, 25-OH, CHL, and DSM), orthosteric inverse agonist (digoxin), and allosteric inverse agonists (MRL-871, FM26, and compound 13) used in this study. indicate the possibility for dual ligand binding in this NR (39, 40, 43) are all allosteric inverse agonists, with varying po- (Fig. 1 B and C) (38, 40). tencies, that reposition H12 into a distinct conformation that Here we reveal the biochemical and structural proof of RORγt prevents coactivator binding (Fig. 1 A and C) (35). dual ligand binding in a variety of orthosteric and allosteric li- gand combinations. Our study also provides a detailed mecha- Dual Ligand Binding Enhances the Stability of the RORγt LBD. Ligand nistic explanation of cooperativity between the two binding sites binding typically improves the thermal stability of NRs via in this NR. Extensive dual ligand binding studies combined with structural and dynamic changes to the protein fold (44, 45). dual ligand protein cocrystallography and molecular dynamics Thermal RORγt protein denaturation assays were performed to (MD) simulations highlight the cooperative binding events and investigate the effect of single and dual ligand binding, as indi- shed light on the underlying molecular mechanism controlling cated by the RORγt melting temperature, Tm (45, 46). In the protein conformation and enhanced dual ligand affinity (Fig. 1A). presence of the 20-OH, the Tm of RORγt increased by 3.6 °C, relative to the DMSO control,