RESUMEN
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/fisiología , Ribonucleasas/fisiología , Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Microscopía por Crioelectrón/métodos , Endonucleasas/metabolismo , Células HEK293 , Humanos , Conformación Molecular , ARN/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/ultraestructura , Ribonucleasas/metabolismo , Ribonucleasas/ultraestructuraRESUMEN
Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/ß5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Remodelación Ósea , Fibronectinas/metabolismo , Integrina alfaV/metabolismo , Osteocitos/metabolismo , Osteólisis/metabolismo , Adipocitos/patología , Animales , Línea Celular Tumoral , Femenino , Fibronectinas/genética , Células HEK293 , Humanos , Integrina alfaV/genética , Ratones , Osteocitos/patología , Osteólisis/genéticaRESUMEN
In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.
Asunto(s)
Proteínas de Unión al ADN , Endopeptidasa Clp , Proteínas de Escherichia coli , Escherichia coli , Proteolisis , Factor sigma , Factores de Transcripción , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Activación Enzimática , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Fosforilación , Dominios Proteicos , Factor sigma/química , Factor sigma/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.
Asunto(s)
Alanina-ARNt Ligasa/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Neuropilina-1/metabolismo , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/genética , Aminoacilación/genética , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/sangre , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Humanos , Linfocitos , Mutación , Neuropilina-1/genética , Cultivo Primario de Células , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Dispersión del Ángulo PequeñoRESUMEN
Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cristalografía por Rayos X , Femenino , Humanos , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
Asunto(s)
Desarrollo de Medicamentos , Colorantes Fluorescentes/farmacología , Ensayos Analíticos de Alto Rendimiento , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Estructura Molecular , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-ActividadRESUMEN
AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the ß-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including ß2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α1ß2γ1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α1ß1γ1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Activadores de Enzimas/química , Proteínas Quinasas Activadas por AMP/metabolismo , Dominio Catalítico , Células Hep G2 , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus Nepenthes are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.
Asunto(s)
Enzimas Inmovilizadas/química , Pepsina A/química , Proteolisis , Animales , Biocatálisis , Deuterio/química , Medición de Intercambio de Deuterio , Espectrometría de Masas , Sarraceniaceae/enzimología , Especificidad por Sustrato , PorcinosRESUMEN
Resistance to endocrine therapies remains a major clinical problem for the treatment of estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor, and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Receptores de Estrógenos/metabolismo , Relación Estructura-ActividadRESUMEN
AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenina/metabolismo , Nucleótidos/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Adenina/química , Sitios de Unión , Humanos , Modelos Moleculares , Nucleótidos/químicaRESUMEN
We sought to develop RORß-selective probe molecules in order to investigate the function of the receptor in vitro and in vivo and its role in the pathophysiology of disease. To accomplish this, we modified a potent dual RORß/RORγ inverse agonist from the primary literature with the goal of improving selectivity for RORß vs RORγ. Truncation of the Western portion of the molecule ablated activity at RORγ and led to a potent series of RORß modulators. Continued exploration of this series investigated alternate replacement cores for the aminothiazole ring. Numerous suitable replacements were found during the course of our SAR investigations and are reported herein.
Asunto(s)
Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Tiofenos/farmacología , Humanos , Espectrometría de Masas/métodosRESUMEN
Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.
RESUMEN
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
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Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Análisis de Varianza , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Deuterio/análisis , Medición de Intercambio de Deuterio/instrumentación , Hidrógeno/análisis , Ligandos , Espectrometría de Masas/instrumentación , Péptidos/análisis , Proteínas/química , Receptores de Calcitriol/química , Reproducibilidad de los ResultadosRESUMEN
Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.
Asunto(s)
Proteínas de la Matriz Extracelular , Sinapsis , Humanos , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas de la Matriz Extracelular/metabolismo , Células Fotorreceptoras/metabolismo , Sinapsis/metabolismoRESUMEN
Jasmonates (JAs) are plant hormones with crucial roles in development and stress resilience. They activate MYC transcription factors by mediating the proteolysis of MYC inhibitors called JAZ proteins. In the absence of JA, JAZ proteins bind and inhibit MYC through the assembly of MYC-JAZ-Novel Interactor of JAZ (NINJA)-TPL repressor complexes. However, JAZ and NINJA are predicted to be largely intrinsically unstructured, which has precluded their experimental structure determination. Through a combination of biochemical, mutational, and biophysical analyses and AlphaFold-derived ColabFold modeling, we characterized JAZ-JAZ and JAZ-NINJA interactions and generated models with detailed, high-confidence domain interfaces. We demonstrate that JAZ, NINJA, and MYC interface domains are dynamic in isolation and become stabilized in a stepwise order upon complex assembly. By contrast, most JAZ and NINJA regions outside of the interfaces remain highly dynamic and cannot be modeled in a single conformation. Our data indicate that the small JAZ Zinc finger expressed in Inflorescence Meristem (ZIM) motif mediates JAZ-JAZ and JAZ-NINJA interactions through separate surfaces, and our data further suggest that NINJA modulates JAZ dimerization. This study advances our understanding of JA signaling by providing insights into the dynamics, interactions, and structure of the JAZ-NINJA core of the JA repressor complex.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Ciclopentanos/metabolismoRESUMEN
Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Ubiquitina-Proteína Ligasas , Proteínas Adaptadoras Transductoras de Señales/química , Microscopía por Crioelectrón , Talidomida/química , Ubiquitina-Proteína Ligasas/química , Dominios Proteicos , Regulación AlostéricaRESUMEN
GPR158 is an orphan G proteincoupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryoelectron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gß5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.