Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

CryoEM structures of adhesion in GPCR CD97: Filling in some of the gaps.

CD97 (ADGRE5) is an adhesion GPCR (aGPCR) that plays crucial roles in the immune system and cancer. In this issue of Cell Chemical Biology, Wang et al.1 present the cryoEM structures of CD97 in complex with G13, Gq, and Gs G protein subtypes, revealing in-depth insight into aGPCR activation and G protein selectivity.

Structural genomics of the human dopamine receptor system.

The dopaminergic system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS); and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinson's Disease (PD) and schizophrenia. Here, we report cryo-EM structures of all five subtypes of human dopamine receptors in complex with G protein and bound to the pan-agonist, rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of rotigotine recognition in different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G protein coupling specificity. Our work provides a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.

Structural insights into the human D1 and D2 dopamine receptor signaling complexes.

The D1- and D2-dopamine receptors (D1R and D2R), which signal through Gs and Gi, respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-Gs and D2R-Gi signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system.

A Structural Understanding of Class B GPCR Selectivity and Activation Revealed.

Ma et al. (2020) and Liang et al. (2020) describe the cryo-EM structures of three class B G protein-coupled receptors (GPCRs) in complex with native peptides and Gs. Their work establishes the structural basis of peptide specificity and a conserved mechanism of receptor activation and G protein coupling for class B GPCRs.

Nanobody-enabled monitoring of kappa opioid receptor states.

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.

Identification of small molecule inhibitors of Interleukin-18.

Interleukin-18 (IL-18) is a pleiotropic pro-inflammatory cytokine belonging to the IL-1 superfamily. IL-18 plays an important role in host innate and adaptive immune defense but its aberrant activities are also associated with inflammatory diseases such as rheumatoid arthritis and Crohn's disease. IL-18 activity is modulated in vivo by its naturally occurring antagonist, IL-18 Binding Protein (IL-18BP). Recent crystal structures of human IL-18 (hIL-18) in complex with its antagonists or cognate receptor(s) have revealed a conserved binding interface on hIL-18. Through virtual screening of the National Cancer Institute Diversity Set II and in vitro competitive ELISA we have identified three compounds (NSC201631, NSC80734, and NSC61610) that disrupt hIL-18 binding to the ectromelia virus IL-18BP. Through cell-based bioassay, we show that NSC80734 inhibits IL-18-induced production of IFN-γ in a dose-dependent manner with an EC50 of ~250 nM. Our results and methodology presented here demonstrate the feasibility of developing small molecule inhibitors that specifically target the rather large interface of IL-18 that is involved in extensive protein-protein interactions with both IL-18BP and its cognate receptor(s). Our data therefore provide the basis for an approach by which small molecules can be identified that modulate IL-18 activity.

Structural basis for antagonizing a host restriction factor by C7 family of poxvirus host-range proteins.

Human sterile alpha motif domain-containing 9 (SAMD9) protein is a host restriction factor for poxviruses, but it can be overcome by some poxvirus host-range proteins that share homology with vaccinia virus C7 protein. To understand the mechanism of action for this important family of host-range factors, we determined the crystal structures of C7 and myxoma virus M64, a C7 family member that is unable to antagonize SAMD9. Despite their different functions and only 23% sequence identity, the two proteins have very similar overall structures, displaying a previously unidentified fold comprised of a compact 12-stranded antiparallel ß-sandwich wrapped in two short α helices. Extensive structure-guided mutagenesis of C7 identified three loops clustered on one edge of the ß sandwich as critical for viral replication and binding with SAMD9. The loops are characterized with functionally important negatively charged, positively charged, and hydrophobic residues, respectively, together forming a unique "three-fingered molecular claw." The key residues of the claw are not conserved in two C7 family members that do not antagonize SAMD9 but are conserved in distantly related C7 family members from four poxvirus genera that infect diverse mammalian species. Indeed, we found that all in the latter group of proteins bind SAMD9. Taken together, our data indicate that diverse mammalian poxviruses use a conserved molecular claw in a C7-like protein to target SAMD9 and overcome host restriction.

Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity.

Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore, interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling. For this reason, plasmodial ADA accepts a wider range of substrates, as it is responsible for deamination of both adenosine and 5'-methylthioadenosine. The latter substrate is not accepted by mammalian ADA homologs. The structural basis for this natural difference in specificity between plasmodial and mammalian ADA has not been well understood. We now report crystal structures of Plasmodium vivax ADA in complex with adenosine, guanosine, and the picomolar inhibitor 2'-deoxycoformycin. These structures highlight a drastic conformational change in plasmodial ADA upon substrate binding that has not been observed for mammalian ADA enzymes. Further, these complexes illuminate the structural basis for the differential substrate specificity and potential drug selectivity between mammalian and parasite enzymes.

Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae.

Type II secretion systems consist of an assembly of 12-15 Gsp proteins responsible for transporting a variety of virulence factors across the outer membrane in several pathogenic bacteria. In Vibrio cholerae, the major virulence factor cholera toxin is secreted by the Eps Type II secretion apparatus consisting of 14 Eps proteins. One of these, EpsE, is a cytoplasmic putative NTPase essential for the functioning of the Eps system and member of the GspE subfamily of Type II secretion ATPases. The crystal structure of a truncated form of EpsE in nucleotide-liganded and unliganded state has been determined, and reveals a two-domain architecture with the four characteristic sequence "boxes" of the GspE subfamily clustering around the nucleotide-binding site of the C-domain. This domain contains two C-terminal subdomains not reported before in this superfamily of NTPases. One of these subdomains contains a four-cysteine motif that appears to be involved in metal binding as revealed by anomalous difference density. The EpsE subunits form a right-handed helical arrangement in the crystal with extensive and conserved contacts between the C and N domains of neighboring subunits. Combining the most conserved interface with the quaternary structure of the C domain in a distant homolog, a hexameric model for EpsE is proposed which may reflect the assembly of this critical protein in the Type II secretion system. The nucleotide ligand contacts both domains in this model. The N2-domain-containing surface of the hexamer appears to be highly conserved in the GspE family and most likely faces the inner membrane interacting with other members of the Eps system.