Author Correction: Structural insights into µ-opioid receptor activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Structural insights into the activation of metabotropic glutamate receptors.

The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online.

Structural insights into the activation of metabotropic glutamate receptors.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.

A topological switch in CFTR modulates channel activity and sensitivity to unfolding.

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the ß-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

Propagation of conformational changes during µ-opioid receptor activation.

µ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the µOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of µOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the µOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the ß2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.

Structural insights into µ-opioid receptor activation.

Activation of the µ-opioid receptor (µOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for µOR activation, here we report a 2.1 Å X-ray crystal structure of the murine µOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the µOR binding pocket are subtle and differ from those observed for agonist-bound structures of the ß2-adrenergic receptor (ß2AR) and the M2 muscarinic receptor. Comparison with active ß2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the µOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Development of Generic G Protein Peptidomimetics Able to Stabilize Active State Gs Protein-Coupled Receptors for Application in Drug Discovery.

G protein-coupled receptors (GPCRs) represent an important group of membrane proteins that play a central role in modern medicine. Unfortunately, conformational promiscuity hampers full therapeutic exploitation of GPCRs, since the largest population of the receptor will adopt a basal conformation, which subsequently challenges screens for agonist drug discovery programs. Herein, we describe a set of peptidomimetics able to mimic the ability of G proteins in stabilizing the active state of the ß2 adrenergic receptor (ß2 AR) and the dopamine 1 receptor (D1R). During fragment-based screening efforts, these (un)constrained peptide analogues of the α5 helix in Gs proteins, were able to identify agonism pre-imprinted fragments for the examined GPCRs, and as such, they behave as a generic tool, enabling an engagement in agonist earmarked discovery programs.

Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor.

The generation of Abs that recognize the native conformation of G protein-coupled receptors can be a challenging task because, like most multimembrane-spanning proteins, they are extremely difficult to purify as native protein. By combining genetic immunization, phage display, and biopanning, we identified two functional monovalent Abs (nanobodies) targeting ChemR23. The two nanobodies (CA4910 and CA5183) were highly specific for the human receptor and bind ChemR23 with moderate affinity. Binding studies also showed that they share a common binding site that overlaps with that of chemerin, the natural ligand of ChemR23. Consistent with these results, we found that the nanobodies were able to antagonize chemerin-induced intracellular calcium increase. The inhibition was partial when chemerin was used as agonist and complete when the chemerin(149-157) nonapeptide was used as agonist. Engineering of a bivalent CA4910 nanobody resulted in a relatively modest increase in affinity but a marked enhancement of efficacy as an antagonist of chemerin induced intracellular calcium mobilization and a much higher potency against the chemerin(149-157) nonapeptide-induced response. We also demonstrated that the fluorescently labeled nanobodies detect ChemR23 on the surface of human primary cell populations as efficiently as a reference mouse mAb and that the bivalent CA4910 nanobody behaves as an efficient antagonist of chemerin-induced chemotaxis of human primary cells. Thus, these nanobodies constitute new tools to study the role of the chemerin/ChemR23 system in physiological and pathological conditions.

Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a ß2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology.

Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies.

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one.

Structure of a bacterial type IV secretion core complex at subnanometre resolution.

Type IV secretion (T4S) systems are able to transport DNAs and/or proteins through the membranes of bacteria. They form large multiprotein complexes consisting of 12 proteins termed VirB1-11 and VirD4. VirB7, 9 and 10 assemble into a 1.07 MegaDalton membrane-spanning core complex (CC), around which all other components assemble. This complex is made of two parts, the O-layer inserted in the outer membrane and the I-layer inserted in the inner membrane. While the structure of the O-layer has been solved by X-ray crystallography, there is no detailed structural information on the I-layer. Using high-resolution cryo-electron microscopy and molecular modelling combined with biochemical approaches, we determined the I-layer structure and located its various components in the electron density. Our results provide new structural insights on the CC, from which the essential features of T4S system mechanisms can be derived.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic ß-cells, which leads to inactivation of the ß-cell proliferating activity of Tmem27. This role of BACE2 in the control of ß-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme ß-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

Structural Basis of µ-Opioid Receptor-Targeting by a Nanobody Antagonist.

The µ-opioid receptor (µOR), a prototypical member of the G protein-coupled receptor (GPCR) family, is the molecular target of opioid analgesics such as morphine and fentanyl. Due to the limitations and severe side effects of currently available opioid drugs, there is considerable interest in developing novel modulators of µOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, are emerging as alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the µOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded µOR ligand and uncover the molecular basis for µOR antagonism by solving the cryo-EM structure of the NbE-µOR complex. NbE displays a unique ligand binding mode and achieves µOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a ß-hairpin loop formed by NbE that deeply inserts into the µOR and centers most binding contacts, we design short peptide analogues that retain µOR antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes novel µOR ligands that can serve as a basis for therapeutic developments.

Accelerating GPCR Drug Discovery With Conformation-Stabilizing VHHs.

The human genome encodes 850 G protein-coupled receptors (GPCRs), half of which are considered potential drug targets. GPCRs transduce extracellular stimuli into a plethora of vital physiological processes. Consequently, GPCRs are an attractive drug target class. This is underlined by the fact that approximately 40% of marketed drugs modulate GPCRs. Intriguingly 60% of non-olfactory GPCRs have no drugs or candidates in clinical development, highlighting the continued potential of GPCRs as drug targets. The discovery of small molecules targeting these GPCRs by conventional high throughput screening (HTS) campaigns is challenging. Although the definition of success varies per company, the success rate of HTS for GPCRs is low compared to other target families (Fujioka and Omori, 2012; Dragovich et al., 2022). Beyond this, GPCR structure determination can be difficult, which often precludes the application of structure-based drug design approaches to arising HTS hits. GPCR structural studies entail the resource-demanding purification of native receptors, which can be challenging as they are inherently unstable when extracted from the lipid matrix. Moreover, GPCRs are flexible molecules that adopt distinct conformations, some of which need to be stabilized if they are to be structurally resolved. The complexity of targeting distinct therapeutically relevant GPCR conformations during the early discovery stages contributes to the high attrition rates for GPCR drug discovery programs. Multiple strategies have been explored in an attempt to stabilize GPCRs in distinct conformations to better understand their pharmacology. This review will focus on the use of camelid-derived immunoglobulin single variable domains (VHHs) that stabilize disease-relevant pharmacological states (termed ConfoBodies by the authors) of GPCRs, as well as GPCR:signal transducer complexes, to accelerate drug discovery. These VHHs are powerful tools for supporting in vitro screening, deconvolution of complex GPCR pharmacology, and structural biology purposes. In order to demonstrate the potential impact of ConfoBodies on translational research, examples are presented of their role in active state screening campaigns and structure-informed rational design to identify de novo chemical space and, subsequently, how such matter can be elaborated into more potent and selective drug candidates with intended pharmacology.

The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

The chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, ß-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving ß-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the ß-arrestin-bias and the atypical nature of CXCR3-B.

A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth.

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.

Improved tumor targeting of anti-epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology.

The approximately 15-kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti-epidermal growth factor receptor (EGFR) Nanobody (alphaEGFR-alphaEGFR) by fusion to a Nanobody unit binding to albumin (alphaAlb). Biodistributions of alphaEGFR-alphaEGFR, alphaEGFR-alphaEGFR-alphaAlb ( approximately 50 kDa), alphaTNF-alphaTNF-alphaAlb (control, binding tumor necrosis factor-alpha), and the approximately 150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with (177)Lu to facilitate quantification. Tumor uptake of (177)Lu-alphaEGFR-alphaEGFR decreased from 5.0 +/- 1.4 to 1.1 +/- 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of alphaAlb. Blood levels of alphaEGFR-alphaEGFR-alphaAlb were 21.2 +/- 2.5, 11.9 +/- 0.6, and 4.0 +/- 1.4 and tumor levels were 19.4 +/- 5.5, 35.2 +/- 7.5, and 28.0 +/- 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 +/- 3.9, 27.1 +/- 7.9, and 25.6 +/- 6.1 %ID/g) and significantly higher than for alphaTNF-alphaTNF-alphaAlb. alphaEGFR-alphaEGFR-alphaAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of alphaEGFR and alphaAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned.

Domain-interface dynamics of CFTR revealed by stabilizing nanobodies.

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.

A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action.

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

Development by Genetic Immunization of Monovalent Antibodies Against Human Vasoactive Intestinal Peptide Receptor 1 (VPAC1), New Innovative, and Versatile Tools to Study VPAC1 Receptor Function.

Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity. They all recognize a similar epitope localized in the extracellular N-terminal domain of the receptor and distinct from the orthosteric binding site. In agreement with binding studies, which showed that the nanobodies did not interfere with VIP binding, all nanobodies were devoid of any functional properties. However, we observed that the binding of two nanobodies was slightly increased in the presence of VPAC1 agonists [vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27)], but decreased in the presence of VPAC1 antagonist. As no evidence of allosteric activity was seen in VIP binding studies nor in functional assays, it is, therefore, possible that the two nanobodies may behave as very weak allosteric modulators of VPAC1, detectable only in some sensitive settings, but not in others. We demonstrated that the fluorescently labeled nanobodies detect VPAC1 on the surface of human leukocytes as efficiently as a reference mouse monoclonal antibody. We also developed a protocol allowing efficient detection of VPAC1 by immunohistochemistry in paraffin-embedded human gastrointestinal tissue sections. Thus, these nanobodies constitute new original tools to further investigate the role of VPAC1 in physiological and pathological conditions.