RESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Although most cases are sporadic and occur later in life, 10-15% of cases are genetic. Loss-of-function mutations in the ring-between-ring E3 ubiquitin ligase parkin, encoded by the PRKN gene, cause autosomal recessive forms of early onset PD. Together with the kinase PINK1, parkin forms a mitochondrial quality control pathway that tags damaged mitochondria for clearance. Under basal conditions, parkin is inhibited and compounds that increase its activity have been proposed as a therapy for PD. Recently, several naturally occurring hyperactive parkin variants were identified, which increased mitophagy in cultured cells. Here, we validate the hyperactivities of these variants in vitro and compare the levels of activity of the variants to those of the wild-type and the well-characterized hyperactive variant, W403A. We also study the effects of mutating the parkin ACT (activating element) on parkin activity in vitro. This work advances our understanding of the pathogenicity of parkin variants and is an important first step in the design of molecules to increase parkin activity.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Mutação , Mitofagia/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Células HEK293RESUMO
Parkin-mediated mitophagy has been studied extensively, but whether mutations in parkin contribute to Parkinson's disease pathogenesis through alternative mechanisms remains unexplored. Using patient-derived dopaminergic neurons, we found that phosphorylation of parkin by Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) at Ser9 leads to activation of parkin in a neuronal-activity-dependent manner. Activated parkin ubiquitinates synaptojanin-1, facilitating its interaction with endophilin A1 and synaptic vesicle recycling. Neurons from PD patients with mutant parkin displayed defective recycling of synaptic vesicles, leading to accumulation of toxic oxidized dopamine that was attenuated by boosting endophilin A1 expression. Notably, combined heterozygous parkin and homozygous PTEN-induced kinase 1 (PINK1) mutations led to earlier disease onset compared with homozygous mutant PINK1 alone, further underscoring a PINK1-independent role for parkin in contributing to disease. Thus, this study identifies a pathway for selective activation of parkin at human dopaminergic synapses and highlights the importance of this mechanism in the pathogenesis of Parkinson's disease.
Assuntos
Neurônios Dopaminérgicos , Doença de Parkinson , Humanos , Neurônios Dopaminérgicos/metabolismo , Mutação , Doença de Parkinson/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Vesículas Sinápticas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Parkinson disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the midbrain. The majority of early onset forms of Parkinson disease are a result of autosomal mutations in PRKN (parkin RBR E3 ubiquitin protein ligase) and PINK1 (PTEN induced kinase 1), which together regulate the clearance of damaged mitochondria from cells through selective autophagy of mitochondria (mitophagy). In a pair of recent papers, we characterized a secondary mechanism of activation of PRKN by PINK1 that is responsible for approximately a quarter of mitophagy in a cellular model. Our deepening understanding of PRKN-PINK1 signaling affords hope for the development of small molecule therapeutics for the treatment of Parkinson disease.
Assuntos
Autofagia , Doença de Parkinson , Humanos , Proteínas Quinases/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Mitocôndrias/metabolismoRESUMO
Parkin and PINK1 regulate a mitochondrial quality control system that is mutated in some early onset forms of Parkinson's disease. Parkin is an E3 ubiquitin ligase and regulated by the mitochondrial kinase PINK1 via a two-step cascade. PINK1 first phosphorylates ubiquitin, which binds a recruitment site on parkin to localize parkin to damaged mitochondria. In the second step, PINK1 phosphorylates parkin on its ubiquitin-like domain (Ubl), which binds a regulatory site to release ubiquitin ligase activity. Recently, an alternative feed-forward mechanism was identified that bypasses the need for parkin phosphorylation through the binding of a second phosphoubiquitin (pUb) molecule. Here, we report the structure of parkin activated through this feed-forward mechanism. The crystal structure of parkin with pUb bound to both the recruitment and regulatory sites reveals the molecular basis for differences in specificity and affinity of the two sites. We use isothermal titration calorimetry measurements to reveal cooperativity between the two binding sites and the role of linker residues for pUbl binding to the regulatory site. The observation of flexibility in the process of parkin activation offers hope for the future design of small molecules for the treatment of Parkinson's disease.
Assuntos
Proteínas Quinases , Ubiquitina-Proteína Ligases/química , Sítios de Ligação , Humanos , Doença de Parkinson/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
Assuntos
Proteínas Quinases , Ubiquitina-Proteína Ligases , Fosforilação/genética , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mutations in Parkin and PINK1 cause early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: (i) thioester intermediate formation on Parkin and (ii) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyses ubiquitin transfer to substrates. We found that His433 in the RING2 domain contributes to the catalysis of acyl transfer. In cells, the mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin (pUb) in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via pUb at these sites.
Assuntos
Proteínas Quinases , Ubiquitina-Proteína Ligases , Mitocôndrias/metabolismo , Mitofagia/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
In the version of this article initially published, RING2 in the schematic to the left in Fig. 1b was mislabeled as RING0. The error has been corrected in the HTML and PDF versions of the article.
RESUMO
Mutations in the ubiquitin ligase parkin are responsible for a familial form of Parkinson's disease. Parkin and the PINK1 kinase regulate a quality-control system for mitochondria. PINK1 phosphorylates ubiquitin on the outer membrane of damaged mitochondria, thus leading to recruitment and activation of parkin via phosphorylation of its ubiquitin-like (Ubl) domain. Here, we describe the mechanism of parkin activation by phosphorylation. The crystal structure of phosphorylated Bactrocera dorsalis (oriental fruit fly) parkin in complex with phosphorylated ubiquitin and an E2 ubiquitin-conjugating enzyme reveals that the key activating step is movement of the Ubl domain and release of the catalytic RING2 domain. Hydrogen/deuterium exchange and NMR experiments with the various intermediates in the activation pathway confirm and extend the interpretation of the crystal structure to mammalian parkin. Our results rationalize previously unexplained Parkinson's disease mutations and the presence of internal linkers that allow large domain movements in parkin.
Assuntos
Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cristalografia por Raios X , Ativação Enzimática , Humanos , Proteínas de Insetos/genética , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Tephritidae/genética , Tephritidae/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.
Assuntos
Ativação Enzimática/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Clonagem Molecular , Cristalização , Humanos , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The Parkinson's disease (PD)-associated proteins, Parkin and PINK1, together comprise a mitochondrial quality control pathway that promotes neuronal survival through autophagy of damaged mitochondria. Three recent studies have found that Parkin recruitment to mitochondria and ubiquitin ligase activity is controlled by the phosphorylation of ubiquitin by PINK1.
RESUMO
Mutations in the PARK2 (parkin) gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING-in-between-RING E3 ubiquitin ligase that exhibits low basal activity. We describe the crystal structure of full-length rat parkin. The structure shows parkin in an autoinhibited state and provides insight into how it is activated. RING0 occludes the ubiquitin acceptor site Cys(431) in RING2, whereas a repressor element of parkin binds RING1 and blocks its E2-binding site. Mutations that disrupted these inhibitory interactions activated parkin both in vitro and in cells. Parkin is neuroprotective, and these findings may provide a structural and mechanistic framework for enhancing parkin activity.
Assuntos
Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Doença de Parkinson , Transtornos Parkinsonianos , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Ratos , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Dedos de ZincoRESUMO
Polo-like kinases (PLKs) are evolutionarily conserved kinases essential for cell cycle regulation. These kinases are characterized by the presence of a C-terminal phosphopeptide-interaction domain, the polo-box domain (PBD). How the functional domains of PLKs work together to promote cell division is not understood. To address this, we performed a genetic screen to identify mutations that independently modulate the kinase and PBD activities of yeast PLK/Cdc5. This screen identified a mutagenic hotspot in the F-helix region of Cdc5 kinase domain that allows one to control kinase activity in vivo. These mutations can be systematically engineered into other major eukaryotic cell cycle kinases to similarly regulate their activity in live cells. Here, using this approach, we show that the kinase activity of Cdc5 can promote the execution of several stages of mitosis independently of PBD activity. In particular, we observe that the activation of Cdc14 and execution of mitotic exit are uniquely sensitive to the modulation of Cdc5 kinase activity. In contrast, PBD-defective mutants are capable of completing mitosis but are unable to maintain spindle pole body integrity. Consistent with this defect, PBD-deficient cells progressively double the size of their genome and ultimately lose genome integrity. Collectively, these results highlight the specific contributions of Cdc5 functional domains to cell division and reveal unexpected mechanisms controlling spindle pole body behavior and genome stability.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica/fisiologia , Mitose/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Western Blotting , Proteínas de Ciclo Celular/isolamento & purificação , Eletroforese , Citometria de Fluxo , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Mitose/genética , Mutação/genética , Fosforilação , Proteínas Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Fuso Acromático/metabolismo , Fuso Acromático/fisiologiaRESUMO
SoxB is an essential component of the bacterial Sox sulfur oxidation pathway. SoxB contains a di-manganese(II) site and is proposed to catalyze the release of sulfate from a protein-bound cysteine S-thiosulfonate. A direct assay for SoxB activity is described. The structure of recombinant Thermus thermophilus SoxB was determined by x-ray crystallography to a resolution of 1.5 A. Structures were also determined for SoxB in complex with the substrate analogue thiosulfate and in complex with the product sulfate. A mechanistic model for SoxB is proposed based on these structures.
Assuntos
Fatores de Transcrição SOXB1/metabolismo , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Citoplasma/metabolismo , Regulação da Expressão Gênica , Hidrólise , Modelos Químicos , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Enxofre/químicaRESUMO
A defining feature of mitosis is the reorganization of chromosomes into highly condensed structures capable of withstanding separation and large-scale intracellular movements. This reorganization is promoted by condensin, an evolutionarily conserved multisubunit ATPase. Here we show, using budding yeast, that condensin is regulated by phosphorylation specifically in anaphase. This phosphorylation depends on several mitotic regulators, and the ultimate effector is the Polo kinase Cdc5. We demonstrate that Cdc5 directly phosphorylates all three regulatory subunits of the condensin complex in vivo and that this causes a hyperactivation of condensin DNA supercoiling activity. Strikingly, abrogation of condensin phosphorylation is incompatible with viability, and cells expressing condensin mutants that have a reduced ability to be phosphorylated in vivo are defective in anaphase-specific chromosome condensation. Our results reveal the existence of a regulatory mechanism essential for the promotion of genome integrity through the stimulation of chromosome condensation in late mitosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromossomos Fúngicos/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Aurora Quinases , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
The bacterial Sox (sulfur oxidizing) system allows the utilization of inorganic sulfur compounds in energy metabolism. Central to this process is the SoxYZ complex that carries the pathway intermediates on a cysteine residue near the C terminus of SoxY. Crystal structures have been determined for Paracoccus pantotrophus SoxYZ with the carrier cysteine in the underivatized state, conjugated to the polysulfide mimic beta-mercaptoethanol, and as the sulfonate adduct pathway intermediate. The carrier cysteine is located on a peptide swinging arm and is bracketed on either side by diglycine dipeptides acting as molecular universal joints. This structure provides a novel solution to the requirement that the cysteine-bound intermediates be able to access and orient themselves within the active sites of multiple partner enzymes. Adjacent to the swinging arm there is a conserved, deep, apolar pocket into which the beta-mercaptoethanol adduct extends. This pocket would be well suited to a role in protecting labile pathway intermediates from adventitious reactions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , Paracoccus pantotrophus/metabolismo , Enxofre/química , Sequência de Aminoácidos , Cristalografia por Raios X , Cisteína/química , Mercaptoetanol/química , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Plasmídeos/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ácidos Sulfônicos/químicaRESUMO
Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Absent in metazoans, this pathway is an attractive target for nontoxic herbicides and drugs. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. Here we characterize YdiB as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. Structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 A resolution, respectively. Both enzymes display a similar architecture with two alpha/beta domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility suggesting that a switch between open and closed conformations occurs upon substrate binding. Sequence analysis and structural comparison led us to propose the catalytic machinery and a model for 3-dehydroshikimate recognition. Furthermore, we discuss the evolutionary and metabolic implications of the presence of two shikimate dehydrogenases in E. coli and other organisms.
Assuntos
Oxirredutases do Álcool/química , Escherichia coli/enzimologia , Adenosina/metabolismo , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia , Dimerização , Difosfatos/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , NADP/metabolismo , Niacinamida/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.
Assuntos
Escherichia coli/enzimologia , Metiltransferases/química , Sequência de Aminoácidos , Domínio Catalítico , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , RNA Ribossômico 23S/química , Homologia de Sequência de AminoácidosRESUMO
The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of the rffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. In Escherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg(2+), refined at 2.6 A to an R-factor of 22.3% (R(free) = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall alpha/beta fold and consists of two domains, a larger nucleotide binding domain (residues 1-115, 222-291) and a smaller sugar-binding domain (116-221), with the active site located at the domain interface. The Mg(2+) ion is coordinated by two conserved aspartates and the alpha-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg(2+) and leads to a proposal for the dynamics along the reaction pathway.
Assuntos
Escherichia coli/enzimologia , Magnésio/química , Nucleotidiltransferases/química , Nucleotídeos de Timina/química , Sítios de Ligação , Catálise , Clonagem Molecular , Cristalografia por Raios X , Dimerização , Escherichia coli/metabolismo , Modelos Moleculares , Nucleotidiltransferases/metabolismo , Conformação ProteicaRESUMO
In Escherichia coli, the pseudouridine synthase RsuA catalyzes formation of pseudouridine (psi) at position 516 in 16S rRNA during assembly of the 30S ribosomal subunit. We have determined the crystal structure of RsuA bound to uracil at 2.0 A resolution and to uridine 5'-monophosphate (UMP) at 2.65 A resolution. RsuA consists of an N-terminal domain connected by an extended linker to the central and C-terminal domains. Uracil and UMP bind in a cleft between the central and C-terminal domains near the catalytic residue Asp 102. The N-terminal domain shows structural similarity to the ribosomal protein S4. Despite only 15% amino acid identity, the other two domains are structurally similar to those of the tRNA-specific psi-synthase TruA, including the position of the catalytic Asp. Our results suggest that all four families of pseudouridine synthases share the same fold of their catalytic domain(s) and uracil-binding site.