Single hormone or synthetic agonist induces Gs/Gi coupling selectivity of EP receptors via distinct binding modes and propagating paths.

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.

Unsaturated bond recognition leads to biased signal in a fatty acid receptor.

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Tethered peptide activation mechanism of the adhesion GPCRs ADGRG2 and ADGRG4.

Adhesion G protein-coupled receptors (aGPCRs) constitute an evolutionarily ancient family of receptors that often undergo autoproteolysis to produce α and ß subunits1-3. A tethered agonism mediated by the 'Stachel sequence' of the ß subunit has been proposed to have central roles in aGPCR activation4-6. Here we present three cryo-electron microscopy structures of aGPCRs coupled to the Gs heterotrimer. Two of these aGPCRs are activated by tethered Stachel sequences-the ADGRG2-ß-Gs complex and the ADGRG4-ß-Gs complex (in which ß indicates the ß subunit of the aGPCR)-and the other is the full-length ADGRG2 in complex with the exogenous ADGRG2 Stachel-sequence-derived peptide agonist IP15 (ADGRG2(FL)-IP15-Gs). The Stachel sequences of both ADGRG2-ß and ADGRG4-ß assume a U shape and insert deeply into the seven-transmembrane bundles. Constituting the FXφφφXφ motif (in which φ represents a hydrophobic residue), five residues of ADGRG2-ß or ADGRG4-ß extend like fingers to mediate binding to the seven-transmembrane domain and activation of the receptor. The structure of the ADGRG2(FL)-IP15-Gs complex reveals the structural basis for the improved binding affinity of IP15 compared with VPM-p15 and indicates that rational design of peptidic agonists could be achieved by exploiting aGPCR-ß structures. By converting the 'finger residues' to acidic residues, we develop a method to generate peptidic antagonists towards several aGPCRs. Collectively, our study provides structural and biochemical insights into the tethered activation mechanism of aGPCRs.

Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade.

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein-coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange-mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

Correction: Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

[This corrects the article DOI: 10.1039/D1SC02653D.].

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and ß-arrestin-1 (ßarr1). Using this strategy, we monitored the subtle but functionally important conformational change of ßarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2.

Arrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

Genetically Encoded Fluorescent Amino Acid for Monitoring Protein Interactions through FRET.

Förster resonance energy transfer (FRET) is a well-established method for studying macromolecular interactions and conformational changes within proteins. Such a method normally uses fluorescent proteins or chemical-labeling methods which are often only accessible to surface-exposed residues and risk-disturbing target protein structures. Here, we demonstrate that the genetic incorporation of a synthetic fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) and natural endogenous fluorophore Tryptophan (Trp) residues of a protein could serve as an efficient FRET pair to monitor protein interactions, using the signaling transducer ß-arrestin-1 as a model system. We used this technology to record the dynamic spectra in both binding and competition experiments of ß-arrestin-1, the contribution of each specific phosphate in ternary complex formation, in a rapid and efficient manner. The determined Kd value for the association between the active arrestin and Fab30 is 0.68 µM in the three-component interaction system. Moreover, we were able to determine the contributions of the site 3 phospho-site and the site 6 phospho-site binding, each contributing to the high affinity ternary complex assembly as 2.7 fold and 15.5 fold, respectively, which were never determined before. These results thus highlighted the potential usage of this new method in measurement of the allosteric-induced enhanced affinity with small amount proteins and in a fast manner and in a complex system. Collectively, our newly developed Trp:Cou FRET system based on genetic expansion technology has extended the molecular toolboxes available for biochemical and structural biology studies.

Crystal structure and catalytic activity of the PPM1K N94K mutant.

Protein Phosphatase Mg2+ /Mn2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.

Allosteric mechanisms underlie GPCR signaling to SH3-domain proteins through arrestin.

Signals from 800 G-protein-coupled receptors (GPCRs) to many SH3 domain-containing proteins (SH3-CPs) regulate important physiological functions. These GPCRs may share a common pathway by signaling to SH3-CPs via agonist-dependent arrestin recruitment rather than through direct interactions. In the present study, 19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with arrestin allosterically regulates the specific conformational states and functional outcomes of remote ß-arrestin 1 proline regions (PRs). The observed NMR chemical shifts of arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment, which was controlled by defined propagation pathways. Moreover, in vitro reconstitution experiments and biophysical results showed that the receptor-arrestin complex promoted SRC kinase activity through an allosteric mechanism. Thus, allosteric regulation of the conformational states of ß-arrestin 1 PRs by GPCRs and the allosteric activation of downstream effectors by arrestin are two important mechanisms underlying GPCR-to-SH3-CP signaling.

[Effect on the motor function of stroke patients by combination of needling at Back-shu point and trunk exercise].

OBJECTIVE: To study the effect on the motor function of stroke patients by combination of needling at Back-shu point and trunk exercise. METHODS: Ninety stroke hemiplegic patients were randomly assigned to the conventional treatment group (as the convention group), the Back-shu point needling group, and the combination of Back-shu point needling and the trunk exercise group, 30 patients in each group. They were treated with the conventional treatment, needling at Back-shu point, and the combination of needling at Back-shu point and trunk exercise. The Fugl-Meyer score (FMA) and modified Barthel index (MBI) score were assessed before treatment and two months after treatment. RESULTS: The three rehabilitation treatment methods were all effective in improving the motor function of stroke hemiplegic patients (P<0.05). The effects in the Back-shu point needling group and the combination of Back-shu point needling and the trunk exercise group were respectively superior to that in the conventional treatment group (P<0.05). The effect in the combination of Back-shu point needling and the trunk exercise group was superior to that in the Back-shu point needling group (P<0.05). CONCLUSION: The combination of Back-shu point needling and the trunk exercise could improve the motor function of stroke hemiplegic patients, and its effect was better than needling at Back-shu point alone.