Members of the proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) play critical roles in processes like hemostasis, thrombosis, development, wound healing, inflammation, and cancer progression. Comprising PAR1-PAR4, these receptors are specifically activated by protease cleavage at their extracellular amino terminus, revealing a 'tethered ligand' that self-activates the receptor. This triggers complex intracellular signaling via G proteins and beta-arrestins, linking external protease signals to cellular functions. To date, direct structural visualization of these ligand-receptor complexes has been limited. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing a shallow and constricted orthosteric binding pocket. Comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix and larger movements of TM7 upon activation. These findings reveal a common activation mechanism for PAR1 and PAR2, highlighting critical residues involved in ligand recognition. Additionally, the structure of PAR2 bound to a pathway selective antagonist, GB88, demonstrates how potent orthosteric engagement can be achieved by a small molecule mimicking the endogenous tethered ligand's interactions.

The proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) include four members, PAR1-PAR4, that play critical roles in hemostasis, thrombosis, embryonic development, wound healing, inflammation, and cancer progression. The PARs share a unique activation mechanism driven by proteinase cleavage at a specific site within the extracellular amino-terminus, exposing a ‘tethered ligand’ that self-activates the receptor. Subsequent activation allows PAR family members to initiate complex intracellular signaling networks via traditional G protein-mediated pathways and beta-arrestin signaling and, in this way, the PARs link extracellular protease signaling molecules to cellular functions. Despite a primary reliance on biochemical studies for understanding tethered ligand recognition, direct structural visualization of these ligand-receptor complexes has been elusive. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing, for the first time, shallow and constricted orthosteric binding pockets and highlighting critical residues involved in ligand recognition and receptor activation. Surprisingly, comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix, a typical signature of GPCR activation, with large movements of TM7 observed upon activation. These insights lead to the identification of a common mechanism for PAR1 and PAR2 activation and provide a structural template for designing novel antagonists targeting the orthosteric binding site, potentially opening new avenues for therapeutic interventions. ### Competing Interest Statement The authors have declared no competing interest.

The OX2 orexin receptor (OX2R) is a highly expressed G protein-coupled receptor (GPCR) in the brain that regulates wakefulness and circadian rhythms in humans. Antagonism of OX2R is a proven therapeutic strategy for insomnia drugs, and agonism of OX2R is a potentially powerful approach for narcolepsy type 1, which is characterized by the death of orexinergic neurons. Until recently, agonism of OX2R had been considered 'undruggable.' We harness cryo-electron microscopy of OX2R-G protein complexes to determine how the first clinically tested OX2R agonist TAK-925 can activate OX2R in a highly selective manner. Two structures of TAK-925-bound OX2R with either a Gq mimetic or Gi reveal that TAK-925 binds at the same site occupied by antagonists, yet interacts with the transmembrane helices to trigger activating microswitches. Our structural and mutagenesis data show that TAK-925's selectivity is mediated by subtle differences between OX1 and OX2 receptor subtypes at the orthosteric pocket. Finally, differences in the polarity of interactions at the G protein binding interfaces help to rationalize OX2R's coupling selectivity for Gq signaling. The mechanisms of TAK-925's binding, activation, and selectivity presented herein will aid in understanding the efficacy of small molecule OX2R agonists for narcolepsy and other circadian disorders.

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

Formylpeptide receptors (FPRs) as G protein-coupled receptors (GPCRs) can recognize formylpeptides derived from pathogens or host cells to function in host defense and cell clearance. In addition, FPRs, especially FPR2, can also recognize other ligands with a large chemical diversity generated at different stages of inflammation to either promote or resolve inflammation in order to maintain a balanced inflammatory response. The mechanism underlying promiscuous ligand recognition and activation of FPRs is not clear. Here we report a cryo-EM structure of FPR2-G i signaling complex with a peptide agonist. The structure reveals a widely open extracellular region with an amphiphilic environment for ligand binding. Together with computational docking and simulation, the structure suggests a molecular basis for the recognition of formylpeptides and a potential mechanism of receptor activation, and reveals conserved and divergent features in G i coupling. Our results provide a basis for understanding the molecular mechanism of the functional promiscuity of FPRs.

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric Gi and Gs, as well as mini-Gs, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric Gi and Gs, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.

The parathyroid hormone receptor-1 (PTH1R) is a class B G protein-coupled receptor central to calcium homeostasis and a therapeutic target for osteoporosis and hypoparathyroidism. Here we report the cryo-electron microscopy structure of human PTH1R bound to a long-acting PTH analog and the stimulatory G protein. The bound peptide adopts an extended helix with its amino terminus inserted deeply into the receptor transmembrane domain (TMD), which leads to partial unwinding of the carboxyl terminus of transmembrane helix 6 and induces a sharp kink at the middle of this helix to allow the receptor to couple with G protein. In contrast to a single TMD structure state, the extracellular domain adopts multiple conformations. These results provide insights into the structural basis and dynamics of PTH binding and receptor activation.

In the PDF version of this Article, owing to a typesetting error, an incorrect figure was used for Extended Data Fig. 5; the correct figure was used in the HTML version. This has been corrected online.

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Aim: Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor (GPCR) signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative Gα i1 β 1 γ 2 G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of Gα i1 β 1 γ 2 . Methods: The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography. Results: The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the Gα-Gβγ interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gα i and the N-termini of both the Gβ and Gγ subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations (T48F and D272F) were designed that completely abolish the GDP binding of the Gi-DN heterotrimer. Conclusion: Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-Gβγ protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.

Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by γ-secretase are associated with Alzheimer's disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by γ-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to γ-secretase. Mutations that alter residues N terminal to the γ-secretase cleavage site Aβ42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the Aβ42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone Aβ42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by γ-secretase. These results suggest a model of γ-secretase substrate recognition, in which the APP region C terminal to the Aβ42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to γ-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins.

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

Mutations in the amyloid precursor protein (APP) gene and the aberrant cleavage of APP by gamma-secretase are associated with Alzheimer's disease (AD). Here we have developed a simple and sensitive cell-based assay to detect APP cleavage by gamma-secretase. Unexpectedly, most familial AD (FAD)-linked APP mutations make APP partially resistant to gamma-secretase. Mutations that alter residues N terminal to the gamma-secretase cleavage site A beta 42 have subtle effects on cleavage efficiency and cleavage-site selectivity. In contrast, mutations that alter residues C terminal to the A beta 42 site reduce cleavage efficiency and dramatically shift cleavage-site specificity toward the aggregation-prone A beta 42. Moreover, mutations that remove positive charge at residue 53 greatly reduce the APP cleavage by gamma-secretase. These results suggest a model of gamma-secretase substrate recognition, in which the APP region C terminal to the A beta 42 site and the positively charged residue at position 53 are the primary determinants for substrate binding and cleavage-site selectivity. We further demonstrate that this model can be extended to gamma-secretase processing of notch receptors, a family of highly conserved cell-surface signaling proteins.

Guanine nucleotide-binding proteins, also named G proteins, play a vital role as molecular switches in signal transduction cascades initiated by the activation of G protein-coupled receptors (GPCRs). Inactive G proteins are heterotrimers that contain an α subunit and a βγ subunit. GDP occupies a binding pocket within the Gα subunit. GPCRs are activated by agonist binding, which induces conformational changes in the GPCRs that allow the association of their corresponding trimeric G proteins and promote the exchange of GDP to GTP within the Gα subunit. GTP binding causes conformational changes within the “switch” regions of the Gα subunit, thus leading to dissociation of the GTP-bound Gα subunit and the Gβγ dimer, which separately interact with downstream effectors. The slow intrinsic GTPase activity of the Gα subunit catalyzes the hydrolysis of GTP to GDP, thereby returning the Gα subunit to an inactivated state and resulting in re-association of the Gα and Gβγ subunits, which terminates the signal transduction. Since the 1990s, high resolution crystal structures of the various stages of the G protein cycle in its active (GTPγS bound)1,2,3, deactivated (GDP bound)4,5, inactive (Gαβγ complex)6,7,8 and downstream effector-bound9,10,11 states have been determined. In combination with extensive physiological and pharmacological studies, these structures shed light on the molecular basis for the function of heterotrimeric G proteins. The first structure of a GPCR-G protein complex, which was released in 2011, was a milestone12 that provided a comprehensive structural basis for transmembrane signaling by GPCRs and G proteins. A recent breakthrough, the structure of the rhodopsin-arrestin complex, provides the basis for understanding the blocking of GPCR coupling to G proteins and arrestin-biased signaling13. These crystallographic studies have revealed three regions with major conformational differences between the GDP- and GTP-bound conformations of Gα, termed the switch I–III regions. Conformational changes in these regions are directly linked to the guanine nucleotide-binding domain, which is formed by five conserved loops called G1–G5 boxes (Figure 1). The G1–G3 boxes play a critical role in coordinating the α-, β-, and γ-phosphate groups and Mg2+, whereas the G4–G5 boxes form the guanine ring-binding site. Multiple steps in the G protein cycle can be interrupted by mutations in the Gα subunit, especially in the guanine nucleotide-binding pocket. These mutations cause the G protein cycle to be blocked, and the mutations have been used to delineate the GPCR-initiated signaling pathways. Several signaling-blocking mutations in the Gα subunit are dominant negative (DN) mutations, because they can block agonist-activated GPCR signaling in the presence of wild type G proteins. To date, there are three known mechanisms for DN mutation: the sequestration of the Gβγ subunits in a complex that is unable to bind activated GPCRs, the sequestration of the activated GPCR by a heterotrimeric complex that cannot exchange GDP and GTP, and the sequestration of the activated GPCR by nucleotide-free Gα14. Because GPCRs are targets for as many as 50% of drugs on the market, DN G proteins have been extensively used to delineate G protein signaling pathways and represent a promising tool to study GPCR-dependent signaling. A large number of mutants with DN phenotypes have been identified for various types of G proteins: S54N in Gαs15, S47C in Gαo16, S48C in Gαi217, G226A in Gαs18, G203T in Gαo19, D273N in Gαo20, and D277N in Gα1121. Moreover, multiple DN Gα mutations have been combined to overcome conditional, nonselective, and weak mutant phenotypes14. For example, a triple Gα mutant, α3β5/G226A/A366S, almost completely inhibits Gα-mediated signaling22, in which the α3β5 mutations replace five G residues in the α3 helix and the α3β5 loops with homologous Gαi residues (N271K, K274D, R280K, T284D, and I285T), which exhibit an enhanced affinity for GPCR. The G226A mutation stabilizes Gβγ binding, impairs the affinity of GTP, and prohibits the GTP-induced conformational change23. The A366S mutation decreases the affinity for GDP and GTPγS, probably by steric hindrance, and restrains Gαs in the empty nucleotide pocket state24. However, most DN phenotypes have been analyzed only functionally, whereas most structural studies have focused on Gα subunits with single mutations rather than G protein heterotrimers8,25,26. In contrast, the precise mechanisms of signal disruption in the context of G protein heterotrimers remain largely elusive. To gain further insight into the DN mechanism of the Gi heterotrimer, we constructed a multiple DN mutant, Gαi G203A/A326S, which corresponds to the G226A and A366S mutations in Gαs, and determined its crystal structure. Static light scattering was used to evaluate the average molecular mass of the Gi-DN heterotrimer. The SEC elution profile, together with light scattering data and the differential refractive index signals, exhibited single peaks, thus indicating that the Gi-DN heterotrimer is highly homogeneous. A molecular mass of 90 kDa was determined by using multi-angle static light scattering, and this measurement was consistent with the calculated mass of the untagged Gi-DN heterotrimer (86.4 kDa) (Figure 2C). We determined the stability of the Gi-DN heterotrimer by using a thermal shift assay. In the absence of exogenous GDP, the Gi-DN heterotrimer presented a lower Tm value (55.3 °C) than wild-type heterotrimer (61.1 °C), thus indicating that the decreased stability of the Gi heterotrimer was caused by G203A/A326S mutations. Additionally, in the presence of 200 μmol/L GDP, the Tm value of the Gi-DN heterotrimer increased from 55.3 °C to 60.0 °C, thus demonstrating that the Gi-DN heterotrimer was further stabilized by GDP (Figure 2D) and retained the capability to bind GDP. The conformation of the Gi-DN heterotrimer resembles that of the GDP-bound wild-type heterotrimer (PDB code 1GP2), with a root mean square deviation (RMSD) of 1.87 Å. Similarly to the wild-type Gi heterotrimer, the C-terminus of the Gγ subunit (E63-S68) and the N-terminus of the Gαi1 subunits (M1-L5) could not be traced in the crystal structure of the Gi-DN heterotrimer. The 3×GSA linker between them also could not be traced, thus indicating that these flexible regions are less likely to affect the structure of the Gi-DN heterotrimer. Compared with the wild type structure, nearly all helices and loops in the Gαi subunit undergo movements accompanied by the subtle displacement of the GαiAH domain away from GαiRas domain. The β propeller, consisting of seven repeat blades of Gβ, also adopts a changed conformation through rotation, and this rotation is probably caused by the rearrangement of the Gαi subunit. Another notable structural difference between the Gi-DN and wild-type heterotrimer is the rotation and displacement of the parallel α-helical coiled coil formed by the amino terminus of the Gβ and Gγ subunits toward the Gαi subunit (Figure 3B). We performed an analysis of the polar and hydrophobic interactions between GDP and the Gi-DN and wild-type heterotrimers by using the Ligplot+ program40. Although the extent of the Van der Waals interactions does not substantially differ, the hydrogen bonding network is rearranged. Compared with wild-type, the Gi-DN heterotrimer forms two extra hydrogen bonds between G1 box residues S47 and T44 and the α- and β-phosphates of GDP but loses four hydrogen bonds between the G4 box residues N269, K270, and D272 and the purine base of GDP (Figure 4B and 4C). The loss of two net hydrogen bonds indicates an attenuated GDP affinity for the Gi-DN heterotrimer. This speculation is supported by previous evidence that the A326S and G203A mutations in the Gαi subunit as well as the homologous mutations G226A and A366S in the Gαs subunit impair the affinity of the Gα subunit for GDP/GTPγS23,24,25,26. To understand why the A326S mutation attenuates GDP binding, we also performed mABP-biased MD simulations of the WTΔHD and DNΔHD GDP-bound Gαi monomer with the helical domain (ΔHD; herein referred to as WT and DN) removed39. Interestingly, significant changes in the conformational dynamics of the A326S-containing G5 box were observed between the WT and DN Gαi. In contrast to the WT simulations, the introduction of a polar residue at A326S decreased the stability of the G5 box in both DN simulations and allowed it to move upward, 10 Å away from its crystallographic position (Figure 4D and 4E). From these simulations, it can be inferred that the A326S mutation destabilizes the binding pocket and thus may also contribute to the lower binding affinity for GDP and GTP. G203, which is located in the G3 box loop, provides critical contacts for the γ-phosphate of GTP. To analyze the influence of the G203A mutation on GTP binding, we built a model of the GTP-bound form of Gi-DN based on the structure of the GTPγS-occupied Gαi A326S mutant protein (PDB code 1BH2)25 by using SWISS-MODEL41. A steric clash between A203 and GTP was observed according to the alignment between the model and 1BH2 (Figure 5A), and the G3 box is rearranged, thus possibly reducing the steric conflict caused by the G203A mutation. In the DN-mutated structure, the backbone amide nitrogen atom of A203 is hydrogen-bonded to the backbone oxygen atom of T181 in the G2 loop, which contributes to the coordination of Mg2+ in the GTP-bound state. This hydrogen bond contact may cause the rearrangement, in which the G2 box moves toward the G3 box (Figure 5B). A326 is located within 3.6 Å of the N7 atom of the guanine ring in the wild type Gi structure (PDB code 1GP2), and the A326S mutation is expected to cause a steric clash between the guanine ring and the slightly larger side chain of the A326S mutation. In the A326S mutant structure, the steric conflict between S326 and the purine base of GDP pushes GDP away from the S326 residue. The serine hydroxyl group is directed into the core of the Ras-like domain and forms a hydrogen bond with N269, thereby causing a conformational change in the residues in the G4 box. The shift of the guanine ring also distorts the perfect geometry of the hydrogen bonds between the carboxylate group of D272 and the two amine groups (N1 and N2) of the guanine base (Figure 5C). The combination of GDP displacement and the rearrangements of residues in the G4 box leads to the weakening of the binding of the A326S mutant to the guanine nucleotide (Figure 5D). The amino-terminal helix of Gαi packs across the D strands of blade 1 and blade 7 of Gβ. In the Gi-DN structure, the αN helix of the Gαi subunit undergoes a rotation and displacement away from blade 7 and toward blade 1 (Figure 6D), thus resulting in the loss of an ionic interaction between the oxygen atom of the D20 side chain in the Gαi subunit and the guanidyl group of R52, which is located in the D strand of blade 7 of the Gβ subunit. This displacement also creates two extra hydrogen bonds between the αN helix of Gαi subunit and the D strand of blade 1 of the Gβ subunit; the R15 guanidyl group is hydrogen bonded to the backbone oxygen atom of V90 and the ND1 atom in the imidazole ring of H91 (Figure 6B and 6C, Table 2). Interface 2 mainly consists of switch II, which is also critical for the binding of the γ-phosphate of GTP and the coordination of Mg2+. Thus, the exchange of GDP for GTP or the hydrolysis of GTP is closely associated with the affinity of the interaction between the Gαi and Gβγ subunits. The switch II displacement of the Gi-DN heterotrimer differs from that of wild type for nearly every residue in switch II. Additionally, the β propeller loops at the Gαi-Gβ interface also adopt a changed conformation. The resultant conformational changes in Gαi and Gβ lead to distinct contacts in interface 2 (Figure 6E–6G). For example, N119 in Gβ extends toward the Gαi subunit and forms two additional hydrogen bonds with the backbone oxygen atom and the hydroxyl oxygen of T182 in switch I, while the hydrogen bond between E186 in the Gαi subunit and W99 in the Gβ subunit is broken by a 1 Å outward movement of the β2 strand (Figure 6E and 6F, Table 2). A complete comparison of the interactions in the Gαi-Gβ interface shows that the Gi-DN heterotrimer has three more pairs of polar contacts than the wild type complex (Table 2), consistently with the increased size of the buried interface, suggesting increased stability of Gα-Gβ interaction in the Gi-DN heterotrimer. The more extensive interactions at the Gα-Gβγ interface have been supported by previous reports. The G203A mutation in Gαi and the homologous G226A mutation in Gαs inhibit the conformational changes that occur after GTP binding and consequently inhibit the release of Gβγ subunits18,26. The penetration of the C-terminal α5 helix of the G protein into the cytoplasmic core of the transmembrane bundle of the activated GPCRs is one of the most striking structural features of the GPCR-G protein interaction. The substitution of A326 with S alters the conformation of the β6-α5 loop, leading to a distinct shift in the α5 helix of the Gα subunit in the Gi-DN heterotrimer, although this shift is smaller in magnitude compared with the one in the receptor-bound state of Gαsβγ in the structure of the β2AR-Gαsβγ complex (Figure 7E). Overall, we conclude that the Gi-DN heterotrimer shares features of the G protein in a receptor-bound state. To reveal the molecular mechanism by which T48F and D272F block GDP binding, we built two mutant protein homology models based on the structure of human Gαi1 G202A (PDB code 3UMS)42. The benzene ring of F48 in the model sterically interferes with the position of the ribose ring (Figure 8G), and F272 almost overlaps with the guanine base, which would prevent GDP access to the pocket (Figure 8H) and further destabilize the Gi-DN heterotrimer. Several dominant negative Gα subunits have been developed to investigate GPCR-G protein signaling pathways. Despite the progress that has been made in understanding the DN effects of G proteins in terms of functional evaluations, the molecular mechanisms of GPCR-dependent signal disruption by DN mutants remains elusive because of the scarcity of the structural evidence for G protein heterotrimers. Here, we solved the structure of G203A and A326S mutated Gαi1β1γ2, and we provide the first report of the DN mechanism caused by these mutations. By comparing the structure of the wild type Gi heterotrimer, we observed subtle conformation changes in the nucleotide-binding pocket that attenuate the affinity for GDP, more extensive contacts in the interface between the Gα subunit and Gβγ-dimer, a distinct displacement of the N-terminal helices of both Gβ and Gγ subunits and a stretch upward of the αC helix in the Gα subunit. In addition, molecular dynamic analysis indicated that the nucleotide-binding pocket of Gα is greatly destabilized by G203A/A326S mutations, thus potentially contributing to its lower binding affinity for GDP and GTP. We speculate that the Gi-DN heterotrimer is characterized by a reduced affinity for both GDP and GTP, leading to a preference for a nucleotide-free state. Thus, the Gi-DN protein fails to accomplish the exchange of GDP for GTP and loses the ability to be activated. The enhanced interaction of the Gα subunit with Gβγ also prevents the dissociation of Gβγ from the heterotrimer, thereby preventing downstream effector activation by the separate subunits. Surprisingly, the Gi-DN heterotrimer adopts the structural features of the G protein in the receptor-bound state, indicating that Gi-DN is in a state that interacts more easily with activated GPCR than wild type G proteins. The Gi-DN heterotrimer attenuates the binding of activated GPCRs with endogenous wild type G proteins in two different ways. First, it sequesters Gβγ with a relatively more potent binding capability, consequently preventing the wild-type Gα from binding to Gβγ, a process indispensable for binding to the activated GPCR. In addition, the Gi-DN heterotrimer preferentially occupies an activated GPCR, thus decreasing the possibility of the stimulation of other wild type G proteins by GPCR and blocking the physiological transducing signal of GPCR. We also found that two extra mutations in the GDP binding pocket have the potential to abolish GDP binding by the Gi-DN heterotrimer. The Gi-DN heterotrimer with mutations T48F and D272F, together with G203A/A326S, did not display a thermal shift after incubation with GDP, thus that these two mutations are likely to abolish GDP binding completely and to lead to a nucleotide-free state of the Gi heterotrimer. This result is consistent with the rationale for the T48F and D272F mutations, which is that the large side chains of the mutated phenyalanine interfere with the binding of the Gαi subunit to the guanine ring. Dominant negative G proteins are frequently used to delineate GPCR-mediated signaling pathways and to exhibit potential usefulness in therapeutic applications43. The dominant negative mechanism revealed in this study provides a solid structural basis for the design of more potent mutants. However, the structural study of the GPCR-G protein complex is crucial to the research of overall GPCR signal pathways; however, the GPCR-G protein complex is difficult to crystallize because of its considerable instability when binding to GDP or GTP. In the structural study of the β2AR-Gs complex, GDP was removed from its binding pocket by apyrase12 to diminish the influence of GDP. As we characterized previously, the DN-Gi mutant appears to stabilize the Gαβγ-receptor complex with a reduced nucleotide affinity. Dominant negative G proteins are expected to favor the stability of the GPCR-G protein complex and to provide an alternative means for the structural study of the GPCR-G protein complex. Ping LIU and Ming-Zhu JIA designed and performed the research and wrote the first draft of the paper. Edward ZHOU solved and analyzed the structure. Parker W DE WAAL and Bradley M DICKSON analyzed the structure and the molecular dynamic analysis. Bo LIU, Li HOU, Yan-ting YIN, and Yan-yong KANG performed the research. Yi SHI and Karsten MELCHER revised and commented on the manuscript. H Eric XU and Yi JIANG supervised the project and wrote the manuscript with contributions from all the authors. This work was supported by grants from the National Natural Science Foundation of China (31300607); the Shanghai Science and Technology Committee (13ZR1447600); the Shanghai Rising-Star Program (14QA1404300); the Outstanding Young Scientist Foundation, Chinese Academy of Sciences (CAS); the Youth Innovation Promotion Association CAS; the SANOFI-SIBS Scholarship; in part by the Jay and Betty Van Andel Foundation, Amway (China); and the Ministry of Science and Technology of China (No 2012CB910403, 2013CB910601, and XDB08020303).

A hallmark of AD is the presence of plaques found between neurons in the brain. These mainly consist of insoluble β-amyloid protein fragments and are thought to be cytotoxic when aggregated. This can lead to neuron death and subsequent loss of memory and perception. The β-amyloid peptide has 40~42 residues and comes from the transmembrane (TM) segment of amyloid precursor protein (APP), which has a large extracellular domain (ECD) and a small intracellular domain (ICD). APP can be processed by two different pathways. It can be cleaved by α-secretase to release the APP ECD. This cleavage blocks production of β-amyloid and reduces plaque buildup. In the second pathway, APP is first cleaved by β-secretase at the extracellular side near the TM segment, and then by γ-secretase within the TM segment. This releases β-amyloid peptides with lengths of 37–43 residues. The two major forms of β-amyloid peptides have 40 (Aβ-40) and 42 residues (Aβ-42) and contain most of the TM segment. These peptides, especially the longer Aβ-42, are hydrophobic and can easily aggregate into large oligomers.

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCF(D3) that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process.