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Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart 1 , 2 . Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits—intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)—to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials 1 – 5 . However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2–DPP6S–KChIP1 dodecamer complex, the Kv4.2–KChIP1 and Kv4.2–DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2–KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1–S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2–KChIP1–DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex. Cryo-electron microscopy structures of the voltage-gated potassium channel Kv4.2 alone and in complex with auxiliary subunits (DPP6S and/or KChIP1) reveal the distinct mechanisms of these two different subunits in modulating channel activity.

G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and β-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side 1 – 3 . However, only antagonist-bound structures are currently available 1 , 4 – 6 , and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of G s and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with G s . The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than β-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor–transducer interface. A new intracellular agonist-binding pocket is identified that is common to many G protein-coupled receptors, which will have implications for the development of biased compounds that target this large group of receptors.

Adenosine receptors play pivotal roles in physiological processes. Adenosine A 3 receptor (A 3 R), the most recently identified adenosine receptor, is expressed in various tissues, exhibiting important roles in neuron, heart, and immune cells, and is often overexpressed in tumors, highlighting the therapeutic potential of A 3 R-selective agents. Recently, we identified RNA-derived N 6 -methyladenosine (m 6 A) as an endogenous agonist for A 3 R, suggesting the relationship between RNA-derived modified adenosine and A 3 R. Despite extensive studies on the other adenosine receptors, the selectivity mechanism of A 3 R, especially for A 3 R-selective agonists such as m 6 A and namodenoson, remained elusive. Here, we identify tRNA-derived N 6 -isopentenyl adenosine (i 6 A) as an A 3 R-selective ligand via screening of modified nucleosides against the adenosine receptors. Like m 6 A, i 6 A is found in the human body and may be an endogenous A 3 R ligand. Our cryo-EM analyses elucidate the A 3 R-G i complexes bound to adenosine, 5’- N -ethylcarboxamidoadenosine (NECA), m 6 A, i 6 A, and namodenoson at overall resolutions of 3.27 Å (adenosine), 2.86 Å (NECA), 3.19 Å (m 6 A), 3.28 Å (i 6 A), and 3.20 Å (namodenoson), suggesting the selectivity and activation mechanism of A 3 R. We further conduct structure-guided engineering of m 6 A-insensitive A 3 R, which may aid future research targeting m 6 A and A 3 R, providing a molecular basis for future drug discovery. Adenosine A 3 receptor (A 3 R) plays important roles in neurons, heart, and immune cells, and is often overexpressed in tumors. Oshima et al. identify tRNA-derived i 6 A as an A 3 R-selective ligand and use cryo-EM to reveal A 3 R’s selectivity and activation mechanisms.

Melatonin receptors (MT 1 and MT 2 ) transduce inhibitory signaling by melatonin ( N -acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT 1 and MT 2 elucidated the basis of ligand entry and recognition, the ligand-induced MT 1 rearrangement leading to G i -coupling remains unclear. Here we report a cryo-EM structure of the human MT 1 –G i signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other G i -coupled receptors, MT 1 exhibits a large outward movement of TM6, which is considered a specific feature of G s -coupled receptors. Structural comparison of G i and G s complexes demonstrated conformational diversity of the C-terminal entry of the G i protein, suggesting loose and variable interactions at the end of the α5 helix of G i protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gα i and provide the basis for the selectivity of G-protein signaling. A cryo-EM structure of the active human melatonin receptor in complex with G i reveals conformational changes upon activation and the molecular basis for G-protein selectivity.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-G i complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34. GPR34 is a GPCR which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative endogenous ligand. Here, authors report two cryo-EM structures of human GPR34-Gi complex with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or its derivative M1.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with either the LysoPS analogue S3E-LysoPS, which contains an ethoxy group at the sn-1 position, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. In both structures, the ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster, and the aromatic fatty acid surrogate of M1 forms stable hydrophobic interactions with the cavity, thus acting as a superagonist. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34, suggesting its short signal duration. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.Competing Interest StatementO.N. is a co-founder and scientific advisor for Curreio. All other authors declare no competing interests.

Adenosine receptors play pivotal roles in physiological processes. Adenosine A receptor (A R), the most recently identified adenosine receptor, is expressed in various tissues, exhibiting important roles in neuron, heart, and immune cells, and is often overexpressed in tumors, highlighting the therapeutic potential of A R-selective agents. Recently, we identified RNA-derived N -methyladenosine (m A) as an endogenous agonist for A R, suggesting the relationship between RNA-derived modified adenosine and A R. Despite extensive studies on the other adenosine receptors, the selectivity mechanism of A R, especially for A R-selective agonists such as m A and namodenoson, remained elusive. Here, we identify tRNA-derived N -isopentenyl adenosine (i A) as an A R-selective ligand via screening of modified nucleosides against the adenosine receptors. Like m A, i A is found in the human body and may be an endogenous A R ligand. Our cryo-EM analyses elucidate the A R-G complexes bound to adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), m A, i A, and namodenoson at overall resolutions of 3.27 Å (adenosine), 2.86 Å (NECA), 3.19 Å (m A), 3.28 Å (i A), and 3.20 Å (namodenoson), suggesting the selectivity and activation mechanism of A R. We further conduct structure-guided engineering of m A-insensitive A R, which may aid future research targeting m A and A R, providing a molecular basis for future drug discovery.

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signals by coupling to various heterotrimeric G proteins. However, the rules governing G protein preference remain largely elusive. MT1 and MT2 are prototypical Gi/o-coupled GPCRs responding to melatonin, a hormone secreted in a circadian manner. We show here that MT1, but not MT2, couples also to Gs proteins in vitro and activates the Gs/cAMP pathway upon long-term melatonin exposure in vivo, mimicking physiological dawn conditions. We solved the cryo–electron microscopy structure of the melatonin-MT1-Gs complex at 3.0Å resolution, which revealed a strikingly distinct binding mode compared to the MT1–Gi complex. The third intracellular loop of MT1 emerges as a key stabilizer for Gs coupling, a feature previously unrecognized. This is the first solved receptor-Gs complex of a primary Gi-coupled GPCRs, providing new structural and functional insights into G protein selectivity and circadian switch of G protein coupling.

Fearful dental patients often cite various dental instruments or procedures as triggers for their dental fear. Thus, visual dental stimuli provoke anxiety. This preliminary study aimed to assess the level of aversion to visual stimuli in dental patients and compare it with that in dentists. A total of 43 dental patients (25 women, 18 men; average age, 29.9 ± 13.3 years; patient group) and 13 dentists (4 women, 9 men; average age, 28.2 ± 2.0 years; dentist group) were included. All participants had previously undergone dental treatment. The dental fear level was assessed using the self-reported Dental Fear Survey (DFS). Thirty-two images associated with dental treatment were prepared and classified into three categories: dental instruments, dental procedures, and the dental environment. All participants rated their level of disgust toward each image on a visual analog scale with scores ranging from 0 to 100. In the patient group, the disgust ratings for tooth extraction, dental drilling, and local anesthesia were >60, which were significantly different from those in the dentist group (Mann-Whitney test, <0.001, =0.001, and =0.001, respectively). The ranking order of the disgust ratings for the 32 images showed significant correlation between the patient and dentist groups (Spearman correlation coefficient, r=0.80, <0.001). In the patient group, the disgust ratings for dental impressions and the interdental brush, dental light, and dental chair were significantly correlated with DFS scores (r=0.61, <0.001; r=0.47, =0.001; r=0.41, =0.006; and r=0.40, =0.008, respectively). This study revealed that patients have more negative feelings toward invasive procedures than dentists. However, a significant correlation was identified between the ranking of aversion-provoking dental stimuli by patients and dentists. Furthermore, the level of aversion to several dental-related items that do not cause pain was correlated with the dental fear level.

Pembrolizumab plus chemotherapy with or without bevacizumab demonstrated prolonged progression‐free survival (PFS) and overall survival (OS) versus chemotherapy in patients with persistent, recurrent, or metastatic cervical cancer in the phase 3, randomized, double‐blind, placebo‐controlled KEYNOTE‐826 study. We report outcomes in patients enrolled in Japan. Patients received pembrolizumab 200 mg or placebo Q3W for up to 35 cycles plus chemotherapy (paclitaxel 175 mg/m2 + cisplatin 50 mg/m2 or carboplatin AUC 5) with or without bevacizumab 15 mg/kg. Dual primary endpoints were PFS per RECIST v1.1 by investigator assessment and OS in the global population; these were evaluated in patients with tumors with PD‐L1 combined positive score (CPS) ≥1, all‐comers, and PD‐L1 CPS ≥10. Fifty‐seven patients from Japan were randomized (pembrolizumab plus chemotherapy, n = 35; placebo plus chemotherapy, n = 22). Pembrolizumab plus chemotherapy improved PFS versus placebo plus chemotherapy in patients with PD‐L1 CPS ≥1 (n = 51; hazard ratio [HR; 95% CI], 0.36 [0.16–0.77]), all‐comers (n = 57; 0.45 [0.22–0.90]), and patients with PD‐L1 CPS ≥10 (n = 25; 0.36 [0.12–1.07]). HRs (95% CI) for OS were 0.38 (0.14–1.01), 0.41 (0.17–1.00), and 0.37 (0.10–1.30), respectively. Incidence of grade 3–5 AEs was 94% in the pembrolizumab group and 100% in the placebo group. Consistent with findings in the global KEYNOTE‐826 study, pembrolizumab plus chemotherapy with or without bevacizumab may prolong survival versus placebo plus chemotherapy with or without bevacizumab and had a manageable safety profile in Japanese patients with persistent, recurrent, or metastatic cervical cancer. Pembrolizumab plus chemotherapy with or without bevacizumab demonstrated prolonged progression‐free survival and overall survival compared with chemotherapy in patients with persistent, recurrent, or metastatic cervical cancer in the phase 3 KEYNOTE‐826 study. In this subset analysis of patients enrolled in Japan in the KEYNOTE‐826 study, pembrolizumab plus chemotherapy with or without bevacizumab was associated with prolonged progression‐free survival and overall survival in this setting.