Christian Bache Billesbølle

Antibody folding and aggregation are major challenges in the development of relevant reagents and therapeutics. Antibodies face a biophysical trade-off; the immense diversity in complementarity-determining regions (CDRs), which is crucial for broad antigen recognition, comes at the cost of folding stability. How CDR sequences influence antibody folding remains poorly understood because of their sequence diversity and lack of large-scale data. Here we develop a high-throughput ‘deep loop profiling’ approach to quantify folding fitness across millions of diverse CDRs. Machine learning models trained on this dataset predict folding propensity directly from sequence and identify interpretable residue-level rules that reveal CDR1 and CDR2 as key folding determinants. Using these insights, we rescue two unstable nanobodies, including an aggregation-prone SARS-CoV-2 binder and a G-protein-coupled receptor-targeting intrabody, and build next-generation synthetic libraries enriched for biophysically optimized nanobodies. This approach provides a scalable framework for understanding and engineering folding competence in antibody-based scaffolds.

/ Summary Paragraph: G protein-coupled receptors (GPCRs) are the largest family of clinically targeted proteins, yet most therapeutics target a narrow subset of structurally well-behaved receptors. The atypical chemokine receptor ACKR3 defies canonical models, displaying broad ligand recognition, high basal activity, and resistance to inhibition. Using engineered nanobodies, cryo-EM, NMR, and structure-guided pharmacology, we uncover an unconventional activation mechanism in ACKR3 that challenges established paradigms of GPCR activation. We find that receptor activity is controlled by changes in extracellular pocket volume rather than conformational rearrangements in conserved microswitches, and an expanded aromatic cluster at the intracellular transducer binding pocket stabilizes the active state. These findings redefine how GPCRs can be modulated and open new strategies for targeting pharmacologically intractable receptors.

Missense variants can have pleiotropic effects on protein function and predicting these effects can be difficult. We performed near-saturation deep mutational scanning of P2RY8, a G-protein-coupled receptor that promotes germinal center B cell confinement. We assayed the effect of each variant on surface expression, migration, and proliferation. We delineated variants that affected both expression and function, affected function independently of expression, and discrepantly affected migration and proliferation. We also used cryo-electron microscopy to determine the structure of activated, ligand-bound P2RY8, providing structural insights into the effects of variants on ligand binding and signal transmission. We applied the deep mutational scanning results to both improve computational variant effect predictions and to characterize the phenotype of germline variants and lymphoma-associated variants. Together, our results demonstrate the power of integrating deep mutational scanning, structure determination, and in silico prediction to advance the understanding of a receptor important in human health.

Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are dominated by a few reaction types, reducing diversity and inevitably leaving many interesting bioactive-like chemotypes unexplored. Here, we investigate the large-scale enumeration and targeted docking of isoquinuclidines. These “natural-product-like” molecules are rare in the current libraries and are functionally congested, making them interesting as receptor probes. Using a modular, four-component reaction scheme, we built and docked a virtual library of over 14.6 million isoquinuclidines against both the µ- and κ -opioid receptors (MOR and KOR, respectively). Synthesis and experimental testing of 18 prioritized compounds found nine ligands with low µM affinities. Structure-based optimization revealed low- and sub- nM antagonists and inverse agonists targeting both receptors. Cryo-electron microscopy (cryoEM) structures illuminate the origins of activity on each target. In mouse behavioral studies, a potent member of the series with joint MOR-antagonist and KOR-inverse-agonist activity reversed morphine-induced analgesia, phenocopying the MOR-selective anti-overdose agent naloxone. Encouragingly, the new molecule induced less severe opioid-induced withdrawal symptoms compared to naloxone during withdrawal precipitation, and did not induce conditioned-place aversion, likely reflecting a reduction of dysphoria due to the compound’s KOR-inverse agonism. The strengths and weaknesses of bespoke library docking, and of docking for opioid receptor polypharmacology, will be considered.

How the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations remains poorly understood. Vertebrate animals perceive odours through G protein-coupled odorant receptors (ORs)¹. In humans, around 400 ORs enable the sense of smell. The OR family comprises two main classes: class I ORs are tuned to carboxylic acids whereas class II ORs, which represent most of the human repertoire, respond to a wide variety of odorants². A fundamental challenge in understanding olfaction is the inability to visualize odorant binding to ORs. Here we uncover molecular properties of odorant–OR interactions by using engineered ORs crafted using a consensus protein design strategy³. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modelling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled the determination of four cryogenic electron microscopy structures of distinct consORs with specific ligand recognition properties. The structure of a class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and generated a homology model of a related member of the human OR51 family with high predictive power. Structures of three class II consORs revealed distinct modes of odorant-binding and activation mechanisms between class I and class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

The lipid prostaglandin E₂ (PGE₂) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.

A central challenge in olfaction is understanding how the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations. Vertebrate animals perceive odors via G protein-coupled odorant receptors (ORs). In humans, ∼400 ORs enable the sense of smell. The OR family is composed of two major classes: Class I ORs are tuned to carboxylic acids while Class II ORs, representing the vast majority of the human repertoire, respond to a wide variety of odorants. How ORs recognize chemically diverse odorants remains poorly understood. A fundamental bottleneck is the inability to visualize odorant binding to ORs. Here, we uncover fundamental molecular properties of odorant-OR interactions by employing engineered ORs crafted using a consensus protein design strategy. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modeling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled four cryoEM structures of distinct consORs with unique ligand recognition properties. The structure of a Class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and yielded a homology model of a related member of the human OR51 family with high predictive power. Structures of three Class II consORs revealed distinct modes of odorant-binding and activation mechanisms between Class I and Class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome^1,2,3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Our sense of smell enables us to navigate a vast space of chemically diverse odor molecules. This task is accomplished by the combinatorial activation of approximately 400 olfactory G protein-coupled receptors (GPCRs) encoded in the human genome ^1–3 . How odorants are recognized by olfactory receptors (ORs) remains mysterious. Here we provide mechanistic insight into how an odorant binds a human olfactory receptor. Using cryogenic electron microscopy (cryo-EM), we determined the structure of active human OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an olfactory receptor. Molecular dynamics simulations demonstrate propionate-induced conformational changes in extracellular loop 3 to activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate OR, providing insight into how this large family of GPCRs enables our olfactory sense.

Thyroid hormones are vital to metabolism, growth and development¹. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR)². Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves’ disease³. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright orientation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody from a Graves’ disease patient selects a similar upright orientation of the extracellular domain. Reorientation of the extracellular domain transduces a conformational change in the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR extracellular domain relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to other G protein-coupled receptors with large extracellular domains.