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KCTD family proteins typically assemble into cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of human KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes to both its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the GTP cyclohydrolase I feedback regulatory protein (GFRP) despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD1 BTB domain fold and identifies an unexpected ion-binding site for future study of KCTD1's function in the ectoderm, neural crest, and kidney.
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High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.
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Electrones , Proteínas de la Membrana , Microscopía por Crioelectrón/métodos , Congelación , Manejo de Especímenes/métodos , Sustancias MacromolecularesRESUMEN
INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.
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Dominio BTB-POZ , Anomalías Craneofaciales , Cara , Humanos , Anomalías Múltiples , Proteínas Co-Represoras/genética , Anomalías Craneofaciales/genética , Displasia Ectodérmica , Cara/anomalías , Mutación Missense/genética , SíndromeRESUMEN
Adenovirus-derived nanoparticles (ADDomer) comprise 60 copies of adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport, and deployment of ADDomer-based therapeutics independent of a cold chain. To expand the scope of ADDomers for new applications, we engineered ADDobodies, representing PBP crown domain, genetically separated from PBP multimerization domain. We inserted heterologous sequences into hyper-variable loops, resulting in monomeric, thermostable ADDobodies expressed at high yields in Escherichia coli. The X-ray structure of an ADDobody prototype validated our design. ADDobodies can be used in ribosome display experiments to select a specific binder against a target, with an enrichment factor of â¼104-fold per round. ADDobodies can be re-converted into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 Å electron cryo-microscopy structure. Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.
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Ingeniería de Proteínas , Sitios de UniónRESUMEN
Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens. Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with in vitro antibody selection and in vivo immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs). In vitro generated neutralizing nanobodies and electron cryo-microscopy established authenticity and accessibility of epitopes displayed by ADDoCoV. Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Vaccinating mice resulted in antibodies cross-reacting with VOCs including Delta and Omicron. Conclusion: Our study elucidates Adenovirus-derived dodecamer (ADDomer)-based nanoparticles for use in active and passive immunization and provides a blueprint for crafting reagents to combat respiratory viral infections.
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UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2's third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B's disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity â¼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with â¼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3's middle-domain elucidates its essential role in NMD.
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Discapacidad Intelectual , Degradación de ARNm Mediada por Codón sin Sentido , Unión Competitiva , Humanos , Discapacidad Intelectual/genética , Mutación , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development.
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Benzamidas/farmacología , Receptor con Dominio Discoidina 1/metabolismo , Receptor con Dominio Discoidina 2/metabolismo , Sulfonamidas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Sitio Alostérico , Animales , Benzamidas/síntesis química , Benzamidas/metabolismo , Línea Celular Tumoral , Receptor con Dominio Discoidina 1/química , Receptor con Dominio Discoidina 2/química , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Microsomas Hepáticos/metabolismo , Unión Proteica , Sulfonamidas/síntesis química , Sulfonamidas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/químicaRESUMEN
Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
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Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Microscopía por Crioelectrón , Células HeLa , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Factores de Terminación de Péptidos/metabolismo , Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína/química , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/efectos de los fármacos , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismoRESUMEN
Receptor-interacting protein kinase 2 (RIPK2) is a key mediator of nucleotide-binding oligomerization domain (NOD) cell signaling that has been implicated in various chronic inflammatory conditions. A new class of RIPK2 kinase/NOD signaling inhibitors based on a 3,5-diphenyl-2-aminopyridine scaffold was developed. Several co-crystal structures of RIPK2â¢inhibitor complexes were analyzed to provide insights into inhibitor selectivity versus the structurally related activin receptor-like kinase 2 (ALK2) demonstrating that the inhibitor sits deeper in the hydrophobic binding pocket of RIPK2 perturbing the orientation of the DFG motif. In addition, the structure-activity relationship study revealed that in addition to anchoring to the hinge and DFG via the 2-aminopyridine and 3-phenylsulfonamide, respectively, appropriate occupancy of the region between the gatekeeper and the αC-helix provided by substituents in the 4- and 5-positions of the 3-phenylsulfonamide were necessary to achieve potent NOD cell signaling inhibition. For example, compound 18t (e.g. CSLP37) displayed potent biochemical RIPK2 kinase inhibition (IC50 = 16 ± 5 nM), >20-fold selectivity versus ALK2 and potent NOD cell signaling inhibition (IC50 = 26 ± 4 nM) in the HEKBlue assay. Finally, in vitro ADME and pharmacokinetic characterization of 18t further supports the prospects of the 3,5-diphenyl-2-aminopyridine scaffold for the generation of in vivo pharmacology probes of RIPK2 kinase and NOD cell signaling functions.
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Aminopiridinas/química , Proteínas Adaptadoras de Señalización NOD/química , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/química , Transducción de Señal/efectos de los fármacos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Inflamación , Relación Estructura-ActividadRESUMEN
Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo-electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.
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Adenoviridae , Mapeo Epitopo/métodos , Epítopos/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/inmunología , Adenoviridae/clasificación , Adenoviridae/genética , Adenoviridae/inmunología , Control de Enfermedades Transmisibles , Enfermedades Transmisibles/etiología , Enfermedades Transmisibles/inmunología , Epítopos/química , Epítopos/genética , Ingeniería Genética , Humanos , Modelos Moleculares , Nanomedicina , Nanotecnología , Conformación Proteica , Relación Estructura-Actividad , Vacunación , Vacunología/métodos , Proteínas Virales/síntesis química , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.
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Biología Computacional , Conformación Proteica , Proteínas/ultraestructura , Arabidopsis/química , Arabidopsis/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/genéticaRESUMEN
RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between ß2 and ß3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling.
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Proteína Adaptadora de Señalización NOD2/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Proteína Adaptadora de Señalización NOD2/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.
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Quinasa de la Caseína I/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de Neoplasias/química , Dominios Proteicos , Quinasa de la Caseína I/química , Quinasa de la Caseína I/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas , Transducción de SeñalRESUMEN
Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro, whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation.
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Degradación de ARNm Mediada por Codón sin Sentido , Biosíntesis de Proteínas , Regiones no Traducidas 3' , Codón sin Sentido , Exones , Humanos , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Transactivadores/metabolismoRESUMEN
Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.
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Compuestos de Fenilurea/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Piridinas/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular , Compuestos de Fenilurea/síntesis química , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Piridinas/síntesis química , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/químicaRESUMEN
Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.
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Proteínas Cullin/química , Proteínas Cullin/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X/métodos , Proteínas Cullin/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.