Frizzleds act as dynamic pharmacological entities.

The Frizzled family of transmembrane receptors (FZD1-10) belongs to the class F of G protein-coupled receptors (GPCRs). FZDs bind to and are activated by Wingless/Int1 (WNT) proteins. The WNT/FZD signaling system regulates crucial aspects of developmental biology and stem-cell regulation. Dysregulation of WNT/FZD communication can lead to developmental defects and diseases such as cancer and fibrosis. Recent insight into the activation mechanisms of FZDs has underlined that protein dynamics and conserved microswitches are essential for FZD-mediated information flow and build the basis for targeting these receptors pharmacologically. In this review, we summarize recent advances in our understanding of FZD activation, and how novel concepts merge and collide with existing dogmas in the field.

Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7.

The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.

Structures of the arginine-vasopressin and oxytocin receptor signaling complexes.

Arginine-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones which share a high sequence and structure homology. These are two cyclic C-terminally amidated nonapeptides with different residues at position 3 and 8. In mammals, AVP and OT exert their multiple biological functions through a specific G protein-coupled receptor family: four receptors are identified, the V1a, V1b, V2 receptors (V1aR, V1bR and V2R) and the OT receptor (OTR). The chemical structure of AVP and OT was elucidated in the early 1950s. Thanks to X-ray crystallography and cryo-electron microscopy, it took however 70 additional years to determine the three-dimensional structures of the OTR and the V2R in complex with their natural agonist ligands and with different signaling partners, G proteins and ß-arrestins. Today, the comparison of the different AVP/OT receptor structures gives structural insights into their orthosteric ligand binding pocket, their molecular mechanisms of activation, and their interfaces with canonical Gs, Gq and ß-arrestin proteins. It also paves the way to future rational drug design and therapeutic compound development. Indeed, agonist, antagonist, biased agonist, or pharmacological chaperone analogues of AVP and OT are promising candidates to regulate different physiological functions and treat several pathologies.

Pathway selectivity in Frizzleds is achieved by conserved micro-switches defining pathway-determining, active conformations.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) paralogs and Smoothened, remains one of the most enigmatic GPCR families. This class mediates signaling predominantly through Disheveled (DVL) or heterotrimeric G proteins. However, the mechanisms underlying pathway selection are elusive. Here we employ a structure-driven mutagenesis approach in combination with an extensive panel of functional signaling readouts to investigate the importance of conserved state-stabilizing residues in FZD5 for signal specification. Similar data were obtained for FZD4 and FZD10 suggesting that our findings can be extrapolated to other members of the FZD family. Comparative molecular dynamics simulations of wild type and selected FZD5 mutants further support the concept that distinct conformational changes in FZDs specify the signal outcome. In conclusion, we find that FZD5 and FZDs in general prefer coupling to DVL rather than heterotrimeric G proteins and that distinct active state micro-switches in the receptor are essential for pathway selection arguing for conformational changes in the receptor protein defining transducer selectivity.

Bringing GPCR Structural Biology to Medical Applications: Insights from Both V2 Vasopressin and Mu-Opioid Receptors.

G-protein coupled receptors (GPCRs) are versatile signaling proteins that regulate key physiological processes in response to a wide variety of extracellular stimuli. The last decade has seen a revolution in the structural biology of clinically important GPCRs. Indeed, the improvement in molecular and biochemical methods to study GPCRs and their transducer complexes, together with advances in cryo-electron microscopy, NMR development, and progress in molecular dynamic simulations, have led to a better understanding of their regulation by ligands of different efficacy and bias. This has also renewed a great interest in GPCR drug discovery, such as finding biased ligands that can either promote or not promote specific regulations. In this review, we focus on two therapeutically relevant GPCR targets, the V2 vasopressin receptor (V2R) and the mu-opioid receptor (µOR), to shed light on the recent structural biology studies and show the impact of this integrative approach on the determination of new potential clinical effective compounds.

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites. However, the scarcity of structural data precludes understanding of how AHR is activated by such diverse compounds. Our 2.85 Å structure of the human indirubin-bound AHR complex with the chaperone Hsp90 and the co-chaperone XAP2, reported herein, reveals a closed conformation Hsp90 dimer with AHR threaded through its lumen and XAP2 serving as a brace. Importantly, we disclose the long-awaited structure of the AHR PAS-B domain revealing a unique organisation of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing structural details of the molecular initiating event leading to AHR activation, our study rationalises almost forty years of biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

Structure of the vasopressin hormone-V2 receptor-ß-arrestin1 ternary complex.

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with ß-arrestin1. It reveals an atypical position of ß-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/ß-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R carboxyl terminus are clearly identified and interact extensively with the ß-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a notable structural variability among GPCR-arrestin signaling complexes.

Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.

Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Cryo-electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex.

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.