RESUMEN

Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. Family D replicative DNA polymerases (PolD), found exclusively in Archaea, contain an unusual RNA polymerase-like catalytic core, and a unique Mre11-like proofreading active site. Here, we present cryo-EM structures of PolD trapped in a proofreading mode, revealing an unanticipated correction mechanism that extends the repertoire of protein domains known to be involved in DNA proofreading. Based on our experimental structures, mutants of PolD were designed and their contribution to mismatch bypass and exonuclease kinetics was determined. This study sheds light on the convergent evolution of structurally distinct families of DNA polymerases, and the domain acquisition and exchange mechanism that occurred during the evolution of the replisome in the three domains of life.

Asunto(s)

ADN Polimerasa Dirigida por ADN , Exonucleasas , Exonucleasas/genética , Exonucleasas/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Replicación del ADN/genética , Dominio Catalítico , Dominios Proteicos

RESUMEN

TsaC/Sua5 family of enzymes catalyzes the first step in the synthesis of N6-threonyl-carbamoyl adenosine (t6A) one of few truly ubiquitous tRNA modifications important for translation accuracy. TsaC is a single domain protein while Sua5 proteins contains a TsaC-like domain and an additional SUA5 domain of unknown function. The emergence of these two proteins and their respective mechanisms for t6A synthesis remain poorly understood. Here, we performed phylogenetic and comparative sequence and structure analysis of TsaC and Sua5 proteins. We confirm that this family is ubiquitous but the co-occurrence of both variants in the same organism is rare and unstable. We further find that obligate symbionts are the only organisms lacking sua5 or tsaC genes. The data suggest that Sua5 was the ancestral version of the enzyme while TsaC arose via loss of the SUA5 domain that occurred multiple times in course of evolution. Multiple losses of one of the two variants in combination with horizontal gene transfers along a large range of phylogenetic distances explains the present day patchy distribution of Sua5 and TsaC. The loss of the SUA5 domain triggered adaptive mutations affecting the substrate binding in TsaC proteins. Finally, we identified atypical Sua5 proteins in Archaeoglobi archaea that seem to be in the process of losing the SUA5 domain through progressive gene erosion. Together, our study uncovers the evolutionary path for emergence of these homologous isofunctional enzymes and lays the groundwork for future experimental studies on the function of TsaC/Sua5 proteins in maintaining faithful translation.

RESUMEN

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.

Asunto(s)

Replicación del ADN , Proteína de Replicación A , Proteína de Replicación A/metabolismo , ADN/metabolismo , ADN de Cadena Simple/genética , Reparación del ADN , Unión Proteica

RESUMEN

To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome's transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).

Asunto(s)

Clonación Molecular , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Escherichia coli/genética , SARS-CoV-2/genética , Proteínas no Estructurales Virales/genética , COVID-19/virología , Clonación Molecular/métodos , ARN Polimerasa Dependiente de ARN de Coronavirus/aislamiento & purificación , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismo

RESUMEN

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2' modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

Asunto(s)

Proteínas Arqueales/genética , ADN de Archaea/genética , ADN Polimerasa Dirigida por ADN/genética , Regulación de la Expresión Génica Arqueal , Genoma Arqueal , Ribonucleótidos/genética , Thermococcus/genética , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Sitios de Unión , Dominio Catalítico , Clonación Molecular , Replicación del ADN , ADN de Archaea/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Expresión Génica , Histidina/química , Histidina/metabolismo , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Thermococcus/enzimología

RESUMEN

GLIC is a bacterial homologue of the pentameric ligand-gated ion channels (pLGICs) that mediate the fast chemical neurotransmission of nerve signalling in eukaryotes. Because the activation and allosteric modulation features are conserved among prokaryotic and eukaryotic pLGICs, GLIC is commonly used as a model to study the allosteric transition and structural pharmacology of pLGICs. It has previously been shown that GLIC is inhibited by some carboxylic acid derivatives. Here, experimental evidence for carboxylate binding to GLIC is provided by solving its X-ray structures with a series of monocarboxylate and dicarboxylate derivatives, and two carboxylate-binding sites are described: (i) the `intersubunit' site that partially overlaps the canonical pLGIC orthosteric site and (ii) the `intrasubunit' vestibular site, which is only occupied by a subset of the described derivatives. While the intersubunit site is widely conserved in all pLGICs, the intrasubunit site is only conserved in cationic eukaryotic pLGICs. This study sheds light on the importance of these two extracellular modulation sites as potential drug targets in pLGICs.

Asunto(s)

Proteínas Bacterianas/metabolismo , Ácidos Carboxílicos/metabolismo , Canales Iónicos Activados por Ligandos/metabolismo , Modelos Moleculares , Preparaciones Farmacéuticas/metabolismo , Sitios de Unión , Cinética , Unión Proteica

RESUMEN

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.

Asunto(s)

ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Archaea , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/química , ADN Polimerasa Dirigida por ADN/genética , Eucariontes , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , Proteínas Recombinantes de Fusión

RESUMEN

DNA and RNA polymerases (DNAP and RNAP) play central roles in genome replication, maintenance and repair, as well as in the expression of genes through their transcription. Multisubunit RNAPs carry out transcription and are represented, without exception, in all cellular life forms as well as in nucleo-cytoplasmic DNA viruses. Since their discovery, multisubunit RNAPs have been the focus of intense structural and functional studies revealing that they all share a well-conserved active-site region called the two-barrel catalytic core. The two-barrel core hosts the polymerase active site, which is located at the interface between two double-psi ß-barrel domains that contribute distinct amino acid residues to the active site in an asymmetrical fashion. Recently, sequencing and structural studies have added a surprising variety of DNA and RNA to the two-barrel superfamily, including the archaeal replicative DNAP (PolD), which extends the family to DNA-dependent DNAPs involved in replication. While all these polymerases share a minimal core that must have been present in their common ancestor, the two-barrel polymerase superfamily now encompasses a remarkable diversity of enzymes, including DNA-dependent RNAPs, RNA-dependent RNAPs, and DNA-dependent DNAPs, which participate in critical biological processes such as DNA transcription, DNA replication, and gene silencing. The present review will discuss both common features and differences among the extended two-barrel polymerase superfamily, focusing on the newly discovered members. Comparing their structures provides insights into the molecular mechanisms evolved by the contemporary two-barrel polymerases to accomplish their different biological functions.

Asunto(s)

Dominio Catalítico , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Evolución Molecular , Conformación Proteica , Subunidades de Proteína/metabolismo

RESUMEN

Replicative DNA polymerases are nano-machines essential to life, which have evolved the ability to copy the genome with high fidelity and high processivity. In contrast with cellular transcriptases and ribosome machines, which evolved by accretion of complexity from a conserved catalytic core, no replicative DNA polymerase is universally conserved. Strikingly, four different families of DNA polymerases have evolved to perform DNA replication in the three domains of life. In Bacteria, the genome is replicated by DNA polymerases belonging to the A- and C-families. In Eukarya, genomic DNA is copied mainly by three distinct replicative DNA polymerases, Polα, Polδ, and Polε, which all belong to the B-family. Matters are more complicated in Archaea, which contain an unusual D-family DNA polymerase (PolD) in addition to PolB, a B-family replicative DNA polymerase that is homologous to the eukaryotic ones. PolD is a heterodimeric DNA polymerase present in all Archaea discovered so far, except Crenarchaea. While PolD is an essential replicative DNA polymerase, it is often underrepresented in the literature when the diversity of DNA polymerases is discussed. Recent structural studies have shown that the structures of both polymerase and proofreading active sites of PolD differ from other structurally characterized DNA polymerases, thereby extending the repertoire of folds known to perform DNA replication. This review aims to provide an updated structural classification of all replicative DNAPs and discuss their evolutionary relationships, both regarding the DNA polymerase and proofreading active sites.

Asunto(s)

Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/clasificación , Archaea , Bacterias , Evolución Biológica , Eucariontes , Conformación Proteica

RESUMEN

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo-electron microscopy (cryo-EM) structure of the heterodimeric DP1-DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1-DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.

Asunto(s)

Proteínas de Unión al ADN/ultraestructura , Factor de Transcripción DP1/ultraestructura , Factores de Transcripción/ultraestructura , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Dominio Catalítico , Microscopía por Crioelectrón/métodos , ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/ultraestructura , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Dominios Proteicos/genética , Subunidades de Proteína/metabolismo , Pyrococcus abyssi/metabolismo , Pyrococcus abyssi/ultraestructura , Factor de Transcripción DP1/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure-function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson-Boltzmann/Debye-Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pKa close to the experimental pH50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pKa at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the highly conserved electrostatic triad R192-D122-D32 and then to Y197-Y119-K248, both located at the extracellular domain-transmembrane domain interface. The second triad controls a cluster of hydrophobic side chains from the M2-M3 loop that is remodeled during the gating transition. We solved 12 crystal structures of GLIC mutants, 6 of them being trapped in an agonist-bound but nonconductive conformation. Combined with previous data, this reveals two branches of a continuous network originating from E35 that reach, independently, the middle transmembrane region of two adjacent subunits. We conclude that GLIC's gating proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/metabolismo , Proteínas Bacterianas/genética , Cianobacterias/química , Cianobacterias/genética , Cinética , Canales Iónicos Activados por Ligandos/genética , Modelos Moleculares , Dominios Proteicos , Protones , Electricidad Estática

RESUMEN

Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

Asunto(s)

Proteínas Bacterianas/química , Gammaproteobacteria/enzimología , Canales Iónicos Activados por Ligandos/química , Regulación Alostérica , Proteínas Bacterianas/antagonistas & inhibidores , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Canales Iónicos Activados por Ligandos/antagonistas & inhibidores , Compuestos de Amonio Cuaternario/química

RESUMEN

Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking. The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and it is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and they provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.

Asunto(s)

Anestésicos Intravenosos/química , Canales Iónicos Activados por Ligandos/química , Modelos Moleculares , Propofol/química , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Anestésicos Intravenosos/farmacología , Animales , Cristalografía por Rayos X , Humanos , Canales Iónicos Activados por Ligandos/genética , Canales Iónicos Activados por Ligandos/metabolismo , Propofol/farmacología , Xenopus laevis

RESUMEN

Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 Å resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi ß-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA transcription and DNA replication within the same protein superfamily. This study documents further the complex evolutionary history of the DNA replication apparatus in different domains of life and proposes a classification of all extant DNAPs.

RESUMEN

Pentameric ligand-gated ion channels have been identified as the principal target of general anesthetics (GA), whose molecular mechanism of action remains poorly understood. Bacterial homologs, such as the Gloeobacter violaceus receptor (GLIC), have been shown to be valid functional models of GA action. The GA bromoform inhibits GLIC at submillimolar concentration. We characterize bromoform binding by crystallography and molecular dynamics (MD) simulations. GLIC's open form structure identified three intra-subunit binding sites. We crystallized the locally closed form with an additional bromoform molecule in the channel pore. We systematically compare binding with the multiple potential sites of allosteric channel regulation in the open, locally closed, and resting forms. MD simulations reveal differential exchange pathways between sites from one form to the other. GAs predominantly access the receptor from the lipid bilayer in all cases. Differential binding affinity among the channel forms is observed; the pore site markedly stabilizes the inactive versus active state.

Asunto(s)

Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/metabolismo , Regulación Alostérica , Anestésicos Generales/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Biológicos , Simulación de Dinámica Molecular , Conformación Proteica , Trihalometanos/metabolismo

RESUMEN

GLIC receptor is a bacterial pentameric ligand-gated ion channel whose action is inhibited by xenon. Xenon has been used in clinical practice as a potent gaseous anaesthetic for decades, but the molecular mechanism of interactions with its integral membrane receptor targets remains poorly understood. Here we characterize by X-ray crystallography the xenon-binding sites within both the open and "locally-closed" (inactive) conformations of GLIC. Major binding sites of xenon, which differ between the two conformations, were identified in three distinct regions that all belong to the trans-membrane domain of GLIC: 1) in an intra-subunit cavity, 2) at the interface between adjacent subunits, and 3) in the pore. The pore site is unique to the locally-closed form where the binding of xenon effectively seals the channel. A putative mechanism of the inhibition of GLIC by xenon is proposed, which might be extended to other pentameric cationic ligand-gated ion channels.

Asunto(s)

Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Cianobacterias/química , Activación del Canal Iónico , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/química , Xenón/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Cianobacterias/metabolismo , Canales Iónicos/metabolismo , Ligandos , Unión Proteica , Estructura Cuaternaria de Proteína , Xenón/metabolismo

RESUMEN

Neurotoxic phospholipases A2 (sPLA2) from snake venoms interact with various protein targets with high specificity and potency. They regulate function of multiple receptors or channels essential to life processes including neuronal or neuromuscular chemoelectric signal transduction. These toxic sPLA2 exhibit high pharmacological potential and determination of PLA2-receptor binding sites represents challenging part in the receptor-channel biochemistry and pharmacology. To investigate the mechanism of interaction of neurotoxic PLA2 with its neuronal receptor at the molecular level, we used as a model crotoxin, a heterodimeric sPLA2 from rattlesnake venom and proton-gated ion channel GLIC, a bacterial homolog of pentameric ligand-gated ion channels. The three-dimensional structures of both partners, crotoxin and GLIC have been solved by X-ray crystallography and production of full-length pentameric GLIC (with ECD and TM domains) is well established. In the present study, for the first time, we demonstrated physical and functional interaction of full-length purified and solubilized GLIC with CB, (PLA2 subunit of crotoxin). We identified GLIC as a new protein target of CB and CB as a new ligand of GLIC, and showed that this non covalent interaction (PLA2-GLIC) involves the extracellular domain of GLIC. We also determined a novel function of CB as an inhibitor of proton-gated ion channel activity. In agreement with conformational changes observed upon formation of the complex, CB appears to be negative allosteric modulator (NAM) of GLIC. Finally, we proposed a possible stoichiometric model for CB - GLIC interaction based on analytical ultracentrifugation.

Asunto(s)

Proteínas Bacterianas/metabolismo , Venenos de Crotálidos/química , Canales Iónicos Activados por Ligandos/metabolismo , Fosfolipasas A2/fisiología , Animales , Cianobacterias/metabolismo , Electrofisiología , Ligandos , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Xenopus laevis

RESUMEN

The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor.

Asunto(s)

Receptores de Glicina/química , Regulación Alostérica/fisiología , Animales , Cristalografía por Rayos X , Drosophila melanogaster , Humanos , Estructura Terciaria de Proteína , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Relación Estructura-Actividad

RESUMEN

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical neurotransmission of nerve signalling in the central and peripheral nervous systems. GLIC is a bacterial homologue of eukaryotic pLGIC, the X-ray structure of which has been determined in three different conformations. GLIC is thus widely used as a model to study the activation and the allosteric transition of this family of receptors. The recently solved high-resolution structure of GLIC (2.4 Šresolution) in the active state revealed two bound acetate molecules in the extracellular domain (ECD). Here, it is shown that these two acetates exactly overlap with known sites of pharmacological importance in pLGICs, and their potential influence on the structure of the open state is studied in detail. Firstly, experimental evidence is presented for the correct assignment of these acetate molecules by using the anomalous dispersion signal of bromoacetate. Secondly, the crystal structure of GLIC in the absence of acetate was solved and it is shown that acetate binding induces local conformational changes that occur in strategic sites of the ECD. It is expected that this acetate-free structure will be useful in future computational studies of the gating transition in GLIC and other pLGICs.

Asunto(s)

Proteínas Bacterianas/química , Canales Iónicos/química , Acetatos/química , Proteínas Bacterianas/genética , Sitios de Unión , Canales Iónicos/genética

RESUMEN

BACKGROUND: Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical transmission of nerve signals in the central and peripheral nervous system. On the functional side, these molecules respond to the binding of a neurotransmitter (glycine, GABA, acetylcholine or 5HT3) in the extracellular domain (ECD) by opening their ionotropic pore in the transmembrane domain (TMD). The response to the neurotransmitter binding can be modulated by several chemical compounds acting at topographically distinct sites, as documented by a large body of literature. Notably, these receptors are the target of several classes of world-wide prescribed drugs, including general anesthetics, smoking cessation aids, anxiolytics, anticonvulsants, muscle relaxants, hypnotics and anti-emetics. On the structural side recent progress has been made on the crystallization of pLGICs in its different allosteric states, especially pLGICs of bacterial origin. Therefore, structure-function relationships can now be discussed at the atomic level for pLGICs. SCOPE OF REVIEW: This review focuses on the crystallographic structure of complexes of pLGICs with a number of ligands of pharmacological interest. First, we review structural data on two key functional aspects of these receptors: the agonist-induced activation and ion transport itself. The molecular understanding of both these functional aspects is important, as they are those that most pharmacological compounds target. Next, we describe modulation sites that have recently been documented by X-ray crystallography. Finally, we propose a simple geometric classification of all these pharmacological sites in pLGICs, based on icosahedrons. MAJOR CONCLUSIONS: This review illustrates the wealth of structural insight gained by comparing all available structures of members of the pLGIC family to rationalize the pharmacology of structurally diverse drugs acting at topographically distinct sites. It will be highlighted how sites that had been described earlier using biochemical techniques can be rationalized using structural data. Surprisingly, the use of icosahedral symmetry allows to link together several modulation sites, in a way that was totally unanticipated. GENERAL SIGNIFICANCE: Overall, understanding the interplay between the different modulation sites at the structural level should help the design of future drugs targeting pLGICs. This article is part of a Special Issue entitled structural biochemistry and biophysics of membrane proteins.

Asunto(s)

Canales Iónicos Activados por Ligandos/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Canales Iónicos Activados por Ligandos/metabolismo , Ligandos , Modelos Moleculares