RESUMO
Trace amine-associated receptor 1 (TAAR1) senses a spectrum of endogenous amine-containing metabolites (EAMs) to mediate diverse psychological functions and is useful for schizophrenia treatment without the side effects of catalepsy. Here, we systematically profiled the signaling properties of TAAR1 activation and present nine structures of TAAR1-Gs/Gq in complex with EAMs, clinical drugs, and synthetic compounds. These structures not only revealed the primary amine recognition pocket (PARP) harboring the conserved acidic D3.32 for conserved amine recognition and "twin" toggle switch for receptor activation but also elucidated that targeting specific residues in the second binding pocket (SBP) allowed modulation of signaling preference. In addition to traditional drug-induced Gs signaling, Gq activation by EAM or synthetic compounds is beneficial to schizophrenia treatment. Our results provided a structural and signaling framework for molecular recognition by TAAR1, which afforded structural templates and signal clues for TAAR1-targeted candidate compounds design.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Aminas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Esquizofrenia/metabolismoRESUMO
Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores de Dopamina D1/metabolismo , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Catecóis/metabolismo , Microscopia Crioeletrônica , Fenoldopam/química , Fenoldopam/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Multimerização Proteica , Receptores de Dopamina D1/química , Receptores de Dopamina D1/ultraestrutura , Receptores de Dopamina D2/metabolismo , Homologia Estrutural de ProteínaRESUMO
Hydroxycarboxylic acid receptor 2 (HCAR2), modulated by endogenous ketone body ß-hydroxybutyrate and exogenous niacin, is a promising therapeutic target for inflammation-related diseases. HCAR2 mediates distinct pathophysiological events by activating Gi/o protein or ß-arrestin effectors. Here, we characterize compound 9n as a Gi-biased allosteric modulator (BAM) of HCAR2 and exhibit anti-inflammatory efficacy in RAW264.7 macrophages via a specific HCAR2-Gi pathway. Furthermore, four structures of HCAR2-Gi complex bound to orthosteric agonists (niacin or monomethyl fumarate), compound 9n, and niacin together with compound 9n simultaneously reveal a common orthosteric site and a unique allosteric site. Combined with functional studies, we decipher the action framework of biased allosteric modulation of compound 9n on the orthosteric site. Moreover, co-administration of compound 9n with orthosteric agonists could enhance anti-inflammatory effects in the mouse model of colitis. Together, our study provides insight to understand the molecular pharmacology of the BAM and facilitates exploring the therapeutic potential of the BAM with orthosteric drugs.
Assuntos
Colite , Receptores Acoplados a Proteínas G , Animais , Camundongos , Regulação Alostérica , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Inflamação/tratamento farmacológico , Corpos Cetônicos , Niacina/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.
Assuntos
Proteínas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Animais , Humanos , Camundongos , Aminas/metabolismo , Anfetamina/metabolismo , Antipsicóticos/química , Antipsicóticos/metabolismo , Sítios de Ligação , Catecolaminas/agonistas , Catecolaminas/química , Catecolaminas/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Ligantes , Simulação de Dinâmica Molecular , Mutação , Polifarmacologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor's orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.
Assuntos
Entropia , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G , Ligantes , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Ligação Proteica , Camundongos , Microscopia Crioeletrônica , Receptores de Canabinoides/metabolismo , Receptores de Canabinoides/química , Sítios de LigaçãoRESUMO
Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.
Assuntos
Microscopia Crioeletrônica , Receptor CB1 de Canabinoide , Transdução de Sinais , Receptor CB1 de Canabinoide/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/química , Animais , Regulação Alostérica/efeitos dos fármacos , Camundongos , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Células HEK293 , Relação Estrutura-Atividade , Dronabinol/farmacologia , Dronabinol/química , Dronabinol/análogos & derivados , Cannabis/química , Cannabis/metabolismoRESUMO
GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.
Assuntos
Inibição Psicológica , Neuralgia , Humanos , Microscopia Crioeletrônica , Ligação CompetitivaRESUMO
Microglia are endogenous immune cells in the brain, and their pyroptosis and phenotype dichotomy are proved to play roles in neurodegenerative diseases. We investigated whether and how hypoxia affected pyroptosis and phenotype polarization in mouse microglia. Primary mouse microglia and BV2 microglia were exposed to hypoxia. Pyroptosis and M1/M2 phenotype were assessed by measuring gasdermin D truncation and M1/M2 surface marker expression. Mechanisms including purinergic ionotropic receptor (P2XR), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) and NOD-like receptor protein 3 (NLRP3) inflammasome were investigated. We reported hypoxia (90% N2, 5% O2 and 5% CO2) induced pyroptosis and promoted M1 phenotype polarization in primary mouse microglia and BV2 microglia, and the effect appeared after 6 h exposure. Although hypoxia (90% N2, 5% O2 and 5% CO2, 6 h) had no effect on P2X1R and P2X7R expression, it increased P2X4R expression and decreased PGC-1α expression. Interestingly, blockade of P2X4R or P2X7R abolished hypoxia-modulated PGC-1α expression, pyroptosis and M1 polarization. PGC-1α overexpression or overactivation alleviated hypoxia-induced pyroptosis and M1 polarization, while PGC-1α knockdown or deactivation promoted pyroptosis and M1 polarization under normoxic situation. Further, hypoxia induced NLRP3 expression and activated caspase-1 and induced the phosphorylation of NF-κB and reduced the phosphorylation of STAT3/6. NLRP3 inhibitor and caspase-1 inhibitor abolished hypoxia-induced pyroptosis, while NF-κB inhibitor and STAT phosphorylation inducer ameliorated hypoxia-induced M1 polarization. In addition, NF-κB activator and STAT3/6 inhibitor caused microglia M1 polarization under normoxic situation. We concluded in cultured mouse microglia, hypoxia may induce pyroptosis via P2XR/PGC-1α/NLRP3/caspase-1 pathway and trigger M1 polarization through P2XR/PGC-1α/NF-κB/STAT3/6 pathway.
Assuntos
Microglia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Piroptose , Transdução de Sinais , Animais , Piroptose/fisiologia , Microglia/metabolismo , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/fisiologia , Diferenciação Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Hipóxia Celular/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Cultivadas , Inflamassomos/metabolismo , Fenótipo , Hipóxia/metabolismoRESUMO
Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.
Assuntos
Simulação de Dinâmica Molecular , Receptor CB1 de Canabinoide , Regulação Alostérica , Sítio Alostérico , Microscopia Crioeletrônica , Receptor CB1 de Canabinoide/genéticaRESUMO
Glucose is essential to the physiological processes of vertebrates. Mammalian physiological stability requires a relatively stable blood glucose level (~5 mM), whereas other vertebrates have greater flexibility in regulating blood glucose (0.5-25 mM). GCGR family receptors play an important role in vertebrate glucose regulation. Here, we examine the evolution of the GCGR family ligand-receptor systems in different species. Comparatively, we discover that the conserved sequences among GCG family ligands lead to the non-specific activation of ligands across species. In particular, we observe that glucagon-like peptide 1 receptor (GLP1R), glucagon-like peptide 2 receptor (GLP2R), and glucagon-like receptor (GCGLR, also called GCRPR) are arbitrarily activated by other members of the ligand family in birds. Moreover, we reveal that Gallus gallus GLP2 (gGLP2) effectively activates mammalian GLP1R and improves glucose tolerance in diabetic mice. Our study has important implications for understanding blood glucose stabilization in vertebrates and demonstrates that gGLP2 may be a potential drug for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Hiperglicemia , Animais , Camundongos , Glicemia , Receptores de Glucagon , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ligantes , Glucose , Hiperglicemia/tratamento farmacológico , Mamíferos , Receptor do Peptídeo Semelhante ao Glucagon 1/genéticaRESUMO
The human cannabinoid G-protein-coupled receptors (GPCRs) CB1 and CB2 mediate the functional responses to the endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) and to the widely consumed plant phytocannabinoid Δ9-tetrahydrocannabinol (THC). The cannabinoid receptors have been the targets of intensive drug discovery efforts, because modulation of these receptors has therapeutic potential to control pain, epilepsy, obesity, and other disorders. Although much progress in understanding the biophysical properties of GPCRs has recently been made, investigations of the molecular mechanisms of the cannabinoids and their receptors have lacked high-resolution structural data. Here we report the use of GPCR engineering and lipidic cubic phase crystallization to determine the structure of the human CB1 receptor bound to the inhibitor taranabant at 2.6-Å resolution. We found that the extracellular surface of CB1, including the highly conserved membrane-proximal N-terminal region, is distinct from those of other lipid-activated GPCRs, forming a critical part of the ligand-binding pocket. Docking studies further demonstrate how this same pocket may accommodate the cannabinoid agonist THC. Our CB1 structure provides an atomic framework for studying cannabinoid receptor function and will aid the design and optimization of therapeutic modulators of the endocannabinoid system.
RESUMO
The CB1 receptor mediates the central nervous system response to cannabinoids, and is a drug target for pain, anxiety and seizures. CB1 also responds to allosteric modulators, which influence cannabinoid binding and efficacy. To understand the mechanism of these compounds, we solved the crystal structure of CB1 with the negative allosteric modulator (NAM) ORG27569 and the agonist CP55940. The structure reveals that the NAM binds to an extrahelical site within the inner leaflet of the membrane, which overlaps with a conserved site of cholesterol interaction in many G protein-coupled receptors (GPCRs). The ternary structure with ORG27569 and CP55940 captures an intermediate state of the receptor, in which aromatic residues at the base of the agonist-binding pocket adopt an inactive conformation despite the large contraction of the orthosteric pocket. The structure illustrates a potential strategy for drug modulation of CB1 and other class A GPCRs.
Assuntos
Receptor CB1 de Canabinoide/metabolismo , Regulação Alostérica , Cristalização , Cicloexanóis/farmacologia , Humanos , Ligação Proteica , Receptor CB1 de Canabinoide/agonistasRESUMO
The NK1 tachykinin G-protein-coupled receptor (GPCR) binds substance P, the first neuropeptide to be discovered in mammals. Through activation of NK1R, substance P modulates a wide variety of physiological and disease processes including nociception, inflammation, and depression. Human NK1R (hNK1R) modulators have shown promise in clinical trials for migraine, depression, and emesis. However, the only currently approved drugs targeting hNK1R are inhibitors for chemotherapy-induced nausea and vomiting (CINV). To better understand the molecular basis of ligand recognition and selectivity, we solved the crystal structure of hNK1R bound to the inhibitor L760735, a close analog of the drug aprepitant. Our crystal structure reveals the basis for antagonist interaction in the deep and narrow orthosteric pocket of the receptor. We used our structure as a template for computational docking and molecular-dynamics simulations to dissect the energetic importance of binding pocket interactions and model the binding of aprepitant. The structure of hNK1R is a valuable tool in the further development of tachykinin receptor modulators for multiple clinical applications.
Assuntos
Morfolinas/metabolismo , Receptores da Neurocinina-1/química , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Morfolinas/química , Ligação Proteica , Conformação Proteica , Substância P/químicaRESUMO
Autophagy is one of the cellular processes that break down cellular components during senescence, starvation, and stress. The susceptibility of plant pollen development to high-temperature (HT) stress is well known, but the involvement of autophagy in HT injury is yet to be clarified. Here, we found that following transfer to 30⯰C, all autophagy-deficient (atg) mutants (atg2-1, 5-1, 7-2, and 10-1) of Arabidopsis thaliana tested displayed visibly impaired pollen development and anther dehiscence. HT-induced male sterility significantly increased in the atg mutants, but the degree of HT-induced obstacles did not change between the wild type (WT) and mutants from the seedling stage to the bolting stage. Cytological analyses showed that 30⯰C promoted autophagy and autolysosome formation in both anther wall cells and microspores in developing anthers of WT, but the atg5-1 mutant did not show completion of tapetum degeneration and microspore maturation. HT upregulated hydrogen peroxide and dehydroascorbate reductase 1 production in both WT and atg5-1 anthers, but the basal levels were already higher in the mutant. HT repressed expression of UNDEAD and its regulator MYB80, which are required for tapetal programmed cell death (PCD) for proper pollen development. Taken together, our results suggest that autophagy functions in tapetum degeneration and pollen development during HT-caused tapetal PCD abortion.
Assuntos
Arabidopsis/metabolismo , Autofagia/fisiologia , Pólen/metabolismo , Apoptose/fisiologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Relacionadas à Autofagia/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Temperatura Alta/efeitos adversos , Infertilidade das Plantas/genética , TemperaturaRESUMO
We report on a high-power picosecond all-fiber Tm-doped fiber amplifier (TDFA) seeded by a gain-switched laser diode (LD) in the 2 µm spectral range. A total average output power of 409 W (304 ps) has been generated at 320 MHz of repetition rate with 10 dB bandwidth of ~48 nm centered at 1970 nm. Over 140 W of spectrally flat supercontinuum (SC) output has been produced at 40 MHz of repetition rate with optimized fiber length. The 10 dB spectral bandwidth was 615 nm, ranging from 1965 to 2580 nm. The prospects for further power scaling of LD seeded ~2 µm picosecond all-fiber sources are discussed.
RESUMO
In this paper, we report on high-power stable nanosecond pulse generation at ~2.1 µm from an integrated Tm-Ho all-fiber master oscillator power amplifier (MOPA) system. A total output power of 128.5 W is generated from the Tm-Ho hybrid MOPA, with an average power of 99.1 W from Ho emission at 2116 nm; the corresponding pulse repetition frequency and pulse width are 161 kHz and 322 ns, respectively, leading to a peak power of 1.91 kW. The Tm-Ho integrated master oscillator is designed to operate at 1980 and 2116 nm, where the former wavelength serves as the pump of the Ho-doped fiber. Stable laser pulses are generated from both the Tm and Ho oscillators owing to mutual modulation of emission from the two lasers. The prospects for further scaling in output power at ~2.1 µm using Tm-Ho integrated MOPA system are discussed.
RESUMO
In this paper, we report on a high-power incoherent beam combination of three Tm-doped fiber amplifiers at 1941 nm based on a 3×1 signal combiner. An output power of 790 W is achieved from the signal combiner with a slope efficiency of 52.2% with respect to the launched pump power and a beam quality factor M2 of 2.7. The beam quality factor is close to the theoretical limit of the 3×1 fiber combiner. The parameters of the amplifier are optimized to increase the laser efficiency. Our analyses show that this structure is sufficient to support kilowatt-level output power while maintaining the high beam quality.
RESUMO
This is an overview of the adaptive optics used in Advanced LIGO (aLIGO), known as the thermal compensation system (TCS). The TCS was designed to minimize thermally induced spatial distortions in the interferometer optical modes and to provide some correction for static curvature errors in the core optics of aLIGO. The TCS is comprised of ring heater actuators, spatially tunable CO2 laser projectors, and Hartmann wavefront sensors. The system meets the requirements of correcting for nominal distortion in aLIGO to a maximum residual error of 5.4 nm rms, weighted across the laser beam, for up to 125 W of laser input power into the interferometer.
RESUMO
Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N(1) methylation of guanine at Position 9 (m(1)G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases.