Quantitative Structural Assessment of Graded Receptor Agonism
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Quantitative structural assessment of graded receptor agonism Jinsai Shanga, Richard Brusta, Patrick R. Griffina,b, Theodore M. Kameneckab, and Douglas J. Kojetina,b,1 aDepartment of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, FL 33458; and bDepartment of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458 Edited by Michael F. Summers, University of Maryland, Baltimore County, Baltimore, MD, and approved September 20, 2019 (received for review June 5, 2019) Ligand–receptor interactions, which are ubiquitous in physiology, cellular transcription factors that recruit chromatin remodeling are described by theoretical models of receptor pharmacology. transcriptional machinery in a ligand-dependent manner to con- Structural evidence for graded efficacy receptor conformations trol gene expression (9). Nuclear receptor agonists, which bind to predicted by receptor theory has been limited but is critical to fully an internal hydrophobic orthosteric pocket within nuclear recep- validate theoretical models. We applied quantitative structure– tor ligand-binding domain (LBD), activate transcription by stabi- function approaches to characterize the effects of structurally sim- lizing structural elements that comprise the activation function-2 ilar and structurally diverse agonists on the conformational ensem- (AF-2) coregulator interaction surface, located adjacent to the γ ble of nuclear receptor peroxisome proliferator-activated receptor ligand-binding pocket, including helix 3, helix 4/5, and a critical γ (PPAR ). For all ligands, agonist functional efficacy is correlated to a regulatory switch element, helix 12. Agonists stabilize an active shift in the conformational ensemble equilibrium from a ground AF-2 surface conformation, which increases the binding affin- state toward an active state, which is detected by NMR spectros- ity for and recruitment of transcriptional coactivator pro- copy but not observed in crystal structures. For the structurally sim- teins that in turn promote chromatin remodeling and increased ilar ligands, ligand potency and affinity are also correlated to transcription. efficacy and conformation, indicating ligand residence times among Although receptor theory as practiced in the membrane re- related analogs may influence receptor conformation and function. ceptor fields is not used in the nuclear receptor field, in principle Our results derived from quantitative graded activity–conformation correlations provide experimental evidence and a platform with the functional endpoints derived from nuclear receptor functional BIOPHYSICS AND assays can be applied to the same pharmacological models of re- which to extend and test theoretical models of receptor pharmacol- COMPUTATIONAL BIOLOGY ogy to more accurately describe and predict ligand-dependent ceptor function. For example, the CTC model (Fig. 1) describes receptor activity. how a receptor exists as a conformational ensemble that fluctuates between resting (R) or active (R*) states capable of binding to nuclear receptor | ligand binding | receptor theory | NMR spectroscopy | ligand (L) and an effector protein or peptide (P). Ligand affinity is X-ray crystallography described by a combination of L + R ↔ LR and L + R* ↔ LR*. The ligand-bound receptor state (LR/LR*) influences effector – eceptor theory has been used to describe the actions of binding (LRP/LR*P) by changing the receptor effector binding Rpharmacological ligands in various forms for nearly a century affinity constant. Ligand potency is related to ligand affinity but (1, 2). The idea of a receptor has evolved from a conceptual “black box” to one founded in the principles of biophysics and Significance allostery (3). The 2-state model of receptor activation (4), which extended the Black/Leff operational model of pharmacological Pharmacological receptor theory models are used to predict agonism (5) with the Monod–Wyman–Changeux model of pro- and interpret how small-molecule ligands impact the function tein allostery (6) to describe the actions of pharmacological re- of cellular receptors. Theoretical models have been modified ceptor ligands, conceptually represents a minimal theoretical and extended over time to more accurately describe relation- model to describe the action of ligands within the context of a ships between ligand affinity and receptor functional. How- binary ligand–receptor complex. More complex models were ever, structural studies are lacking that describe the influence subsequently developed that accounted for improved under- of receptor conformation, or shape, as predicted by these standing of receptor functions. The extended ternary complex models. Using NMR spectroscopy, we detected how the nuclear (ETC) model describes how the receptor–ligand complex influ- receptor transcription factor peroxisome proliferator-activated ences interaction with an effector protein or a signaling pathway receptor γ (PPARγ) structurally responds to ligand binding. We (7), whereas the cubic ternary complex (CTC) model extends the observed a correlation between ligand affinity, function, and ETC model to account for receptor–effector interactions in the receptor conformation for chemically similar ligands but not absence of ligand (8). These and other theoretical receptor chemical diverse ligands, revealing a potential limitation of models could be further improved with a more comprehensive theoretical receptor models. experimental understanding of how ligands affect receptor struc- Author contributions: J.S. and D.J.K. designed research; J.S., R.B., and D.J.K. performed ture and function. research; P.R.G. and T.M.K. contributed new reagents/analytic tools; J.S. and D.J.K. ana- Applying theoretical receptor models to graded activity dose- lyzed data; and J.S. and D.J.K. wrote the paper. responsive pharmacological data are common in studies of mem- The authors declare no competing interest. – brane receptors, including G-protein coupled receptors (GPCRs), This article is a PNAS Direct Submission. ligand-gated ion channels, and enzyme-linked receptors (2). These Published under the PNAS license. receptors bind extracellular ligands and transduce signals across Data deposition: The crystal structures generated in the current study have been depos- the cell membrane via conformational rearrangement of the in- ited in the Protein Data Bank (PDB), http://www.wwpdb.org/ (ID codes 6DGL, 6DGO, tracellular portion of the membrane receptor to affect various 6DGQ, 6DGR, 6O67, and 6O68). downstream signaling pathways, the activities of which can be 1To whom correspondence may be addressed. Email: [email protected]. measured in cellular assays and applied to pharmacological models This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of receptor function. Conceptually, the principles of receptor 1073/pnas.1909016116/-/DCSupplemental. theory also apply to nuclear receptors, a superfamily of intra- First published October 14, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1909016116 PNAS | October 29, 2019 | vol. 116 | no. 44 | 22179–22188 Downloaded by guest on September 24, 2021 approaches can predict ligand functional efficacy and assess R*P LR*P theoretical models of receptor function. Results Graded Potency and Functional Efficacy within a Structurally Related Series of PPARγ Agonists. We assembled a series of 10 thiazolidinedione (TZD) PPARγ agonists (Fig. 2A) that include several US Food and Drug Administration-approved antidiabetic drugs (13). All ligands within series contain the conserved TZD head group connected R* LR* by a linker to a central aromatic moiety and a variable tail group. The linker in all but one of the ligands is a flexible, saturated methylene group that links the TZD head group to a central aromatic moiety; RP LRP CAY10638 contains an unsaturated linker, which restricts the mobility of the TZD head group. The central aromatic moieties mostly comprise phenyl moieties with the exception of naphthalene and benzothiophene moieties in netoglitazone and edaglitazone, respectively. In contrast to these relatively conservative changes near the TZD head group, the series encompasses a variety of tail moieties extended from the central aromatic core. Agonists increase PPARγ-mediated transcription by enhanc- RLRing binding of transcriptional coactivator proteins, such as TRAP220, also known as MED1 or DRIP205 (14). We assessed Fig. 1. The cubic ternary complex (CTC) model. This theoretical model the activities of the TZD series in 2 quantitative biochemical schematic describes the relationship the receptor resting (R) and active (R*) assays. We used a time-resolved fluorescence resonance energy state when complexed to ligand (L), an effector protein or peptide (P), or transfer (TR-FRET) biochemical assay that measures the ligand- both. In the absence of an effector, the apo-state is represented by R and R*, γ and the ligand-bound state by LR and LR*, also known as the 2-state model dependent change in the interaction between the PPAR LBD of receptor activation. and a peptide derived from the TRAP220 coactivator (Fig. 2B) containing an “LXXLL” nuclear receptor interaction motif (15). In the TR-FRET coactivator recruitment assay, differences in describes the concentration of ligand required to elicit the func- the overall TR-FRET ratio assay window span, which we refer tional response (efficacy). Assuming that the ligand-free receptor to as TR-FRET functional efficacy, relates to the relative can interact