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Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

Co-crystal structures of GLIC, a bacterial ligand-gated ion channel, in complex with monocarboxylate and dicarboxylate derivatives are reported. It is shown that binding occurs at two pharmacological sites in the extracellular domain, which is in agreement with the reported effect of some carboxylates as allosteric modulators of GLIC., 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.

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.

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., Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. Here, the authors present a cryo-EM structure of the DNA-bound PolD–PCNA complex from Pyrococcus abyssi to reveal the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA.

SummaryReplicative 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 fromPyrococcus abyssiat 3.77Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography and protein-protein interaction measurements - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA with an unprecedented level of detail. PolD recruits PCNAviaa 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 phase and a processive phase during replication.

International audience; 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 β-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.

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.

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 pK a close to the experimental pH 50 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 pK a 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.

International audience; 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.

Summary: 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. : General anesthetics can both potentiate and inhibit pentameric ligand-gated ion channels, yet the structural basis for their effects remains unclear. Ten crystal structures and associated electrophysiology data by Fourati et al. point to a unified allosteric model for positive and negative modulation, providing a framework for structure-inspired drug design. Keywords: ion channels, general anesthetics, x-ray crystallography, electrophysiology, allostery

International audience; 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.

International audience; 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.

International audience; 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.

International audience; Pentameric ligand-gated ion channels mediate signal transduction through conformational transitions between closed-pore and open-pore states. To stabilize a closed conformation of GLIC, a bacterial proton-gated homolog from Gloeobacter violaceus whose open structure is known, we separately generated either four cross-links or two single mutations. We found all six mutants to be in the same 'locally closed' conformation using X-ray crystallography, sharing most of the features of the open form but showing a locally closed pore as a result of a concerted bending of all of its M2 helices. The mutants adopt several variant conformations of the M2-M3 loop, and in all cases an interacting lipid that is observed in the open form disappears. A single cross-linked mutant is functional, according to electrophysiology, and the locally closed structure of this mutant indicates that it has an increased flexibility. Further cross-linking, accessibility and molecular dynamics data suggest that the locally closed form is a functionally relevant conformation that occurs during allosteric gating transitions.

13 páginas, 7 figuras -- PAGS nros. 8187-8199, Núñez-Ramírez, Rafael et al., The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA–DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA–DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes., The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication, Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.); the ‘Red Temática de Investigación Cooperativa en Cáncer (RTICC)’ from the ‘Instituto de Salud Carlos III’ (RD06/0020/1001 to O.L.); the Human Frontiers Science Program (RGP39/2008 to O.L. and E.N.); a Wellcome Trust Senior Fellowship award in Basic Biomedical Sciences (to L.P.); a FPI fellowship from the Spanish Ministry of Science and Innovation to MAR-C; a JAE-DOC contract of the ‘Consejo Superior de Investigaciones Científicas (CSIC)’ and a ‘Juan de la Cierva’ contract from the Spanish Ministry of Science to BGA; EN is a Howard Hughes Medical Institute Investigator. Funding for open access charge: Spanish Ministry of Science and Innovation (SAF2008-00451 to O.L.)

Structural Details of an Allosteric Mechanism for Bimodal Anesthetic Modulation of Pentameric Ligand-Gated Ion Channels

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.

International audience; 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 Å resolution) 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.

International audience; Pentameric ligand-gated ion channels mediate fast chemical transmission of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformations at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric ligand-gated ion channel. The large structural variability in the neutral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the intrinsic fluctuations in this state, which are found to prefigure the transition to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motion, and the pore in the transmembrane domain (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer interacting across adjacent subunits. On the tertiary level, detachment of inner and outer β sheets in the ECD reshapes two essential cavities at the ECD-ECD and ECD-TMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent cation binding site in other pentameric ligand-gated ion channels. In addition, a different crystal form reveals that the locally closed and open conformations coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transition pathway.

Sauguet et al. recently solved the structure of the pentameric ligand-gated ion channel GLIC in complex with the general anesthetic bromoform [1]. This structure highlights three binding sites in an intra-subunit cavity close to the propofol and desflurane binding sites previously described by Nury et al. [2]. Additionally, a F238A (F14'A) mutant reveals a supplementary inter-subunit binding site.In this work we characterize these and additional sites by computational methods. We combine several approaches to address three key questions: (i) are the crystal binding sites spontaneously accessible? (ii) can bromoform travel from one site to another? (iii) what is the bromoform affinity for each binding site? Molecular dynamics (MD) simulations of flooding the receptor with bromoform recover most of the experimentally observed sites, with a modulated occupancy between the open and the locally closed conformations. Hundreds of short MD simulations were carried out to extensively explore the binding pockets, providing data on possible routes connecting them. These simulations furthermore highlight residues that may play key roles in controlling the interaction between anesthetic and receptor molecules. Free energy of binding calculations indicate significant affinity for all crystallographic binding sites in open and locally closed conformations, in some cases modulated by pH.[1] Sauguet et al. 2013, Nature communications. 4:1697.[2] Nury et al. 2011, Nature. 469:428-31.

Pentameric ligand-gated ion channels (pLGIC) mediate fast chemical transmission of nerve signals. The structure of GLIC, a bacterial proton-gated homolog, has been established in its open and Locally Closed (LC) conformations at acidic pH. Here we report its crystal structure at neutral pH, revealing for the first time the two end-points of the gating mechanism in the same pLGIC using X-ray structures. The structural variability in the neutral pH structure observed in the crystal due to the presence of four copies in the asymmetric unit can be used to analyse the intrinsic fluctuations in this state. It is found that they have a marked tendency to occur in the direction of the closed-to-open transition. In the extra-cellular domain (ECD), an important quaternary change is observed, involving both a twist and a blooming motion, while the transmembrane channel (TMD) is closed in an LC-manner. On the tertiary level, detachment of inner and outer beta-sheets in the ECD reshapes two essential cavities at the ECD-ECD and ECD-TMD interfaces. The first one is the ligand-binding cavity, the other is a new one that matches a known Ca++ or Zn++ binding site in some pLGICs. These results shed new light on the allosteric transitions of pLGIC and their pharmacology. In addition, a plausible pathway between the two forms is presented and discussed.

International audience; Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol.

Alcohol consumption produces a variety of undesirable behavioural and physiological effects in animals and humans and can eventually lead to addiction. Converging evidences suggest that its molecular mechanisms of action involve specific protein targets. Among these, pentameric ligand-gated ion channels (pLGICs) and especially GABA-A Receptor have been shown to be one of the main targets of ethanol in the central nervous system. Here we report the first atomic-resolution structure at 2.8 A of ethanol bound to a member of the pLGIC family, the pH-gated prokaryotic homolog GLIC variant F14'A. This GLIC variant is potentiated by concentrations of ethanol similar to the ones effective in the vertebrate Glycine and GABA-A receptors (R. Howard et al., 2012). Comparison the ethanol-bound and apo structures of GLIC F14'A gives a rational and simple explanation to the potentiating effect of ethanol on these receptors by stabilizing the open form. Multiple-sequence alignments and homology structure models suggest that the ethanol binding-pocket identified in GLIC is also present in human Glycine and GABA-A receptors.

International audience; Orthologs of the pentameric receptor channels that mediate fast synaptic transmission in the central and peripheral nervous systems have been found in several bacterial species and in a single archaea genus. Recent X-ray structures of bacterial and invertebrate pentameric receptors point to a striking conservation of the structural features within the whole family, even between distant prokaryotic and eukaryotic members. These structural data reveal general principles of molecular organization that allow allosteric membrane proteins to mediate chemoelectric transduction. Notably, several conformations have been solved, including open and closed channels with distinct global tertiary and quaternary structure. The data reveal features of the ion channel architecture and of diverse categories of binding sites, such as those that bind orthosteric ligands, including neurotransmitters, and those that bind allosteric modulators, such as general anesthetics, ivermectin, or lipids. In this review, we summarize the most recent data, discuss insights into the mechanism of action in these systems, and elaborate on newly opened avenues for drug design.

Cys-loop receptors mediate synaptic transmission in the nervous system. These proteins bind neurotransmitters thereby promoting the opening of an intrinsic ionic transmembrane channel that gives birth to electrical signal. This global allosteric transition between the resting and active (open-channel) state is followed by a slower desensitization process. Structural data on the cys-loop receptor family was recently moved to atomic resolution thanks to the crystallization of bacterial homologs. The ELIC homolog structure was resolved in a closed-channel conformation, which is thought to represent a resting state. The structure of the GLIC homolog, solved in the presence of its agonist proton shows an open-channel conformation and is thought to represent an active state. Comparison of the two structures suggest that during activation extracellular agonist binding is transduced into the channel opening notably through reorganization of interdomain loops β1β2 and M2M3.To check this GLIC/ELIC model, we designed four double cysteine mutants of GLIC which cross-linking is predicted to differentially stabilize the resting (ELIC) and active (GLIC) states. Those mutants were characterized by two-electrode voltage-clamp electrophysiology upon expression in Xenopus oocytes. All cross-linked mutants were spontaneously bridged : three of them were non-activatable, and return to a wild-type behaviour after DTT treatment, while the one predicted to be the closer to the GLIC structure was activatable, whatever the redox environment. This suggests that the GLIC conformation in the crystals better represents the native conformations than the ELIC one.Bocquet et al. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature (2009) pp. 4Hilf et al. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature (2008) vol. 452 (7185) pp. 375-9

International audience; Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

Background DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood. Methodology/Principal Findings Here we report the crystal structure of the yeast PriL-CTD at 1.55 A resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase. Conclusion/Significance Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.

International audience; Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).

Pentameric ligand-gated ion channels (pLGIC) mediate fast chemical transmission of nerve signals. The structure of GLIC, a bacterial proton-gated homolog, has been established in its open and Locally Closed (LC) conformations at acidic pH. Here we report a new crystal form where the LC and open conformations coexist as discrete forms at acidic pH. These results, together with site-directed mutagenesis and physiological recordings, shed new light on the allosteric transitions of pLGIC and their pharmacology.

Alcohol and Anesthetic Binding to Pentameric Ligand-Gated Ion Channels Revealed in a Prokaryotic Model System

The Poisson-Boltzmann (PB) formalism is one of the most popular approaches to modeling the solvation of molecules. It assumes a continuum model for water, leading to a dielectric permittivity that depends on regions of space (inside or outside the solute). In contrast, the dipolar Poisson-Boltzmann-Langevin (DPBL) formalism follows a grid-based approach where the solvent is represented as a collection of freely orientable dipoles with nonuniform concentration [1]. This leads to a nonlinear permittivity function that depends both on the position and on the local electric field at that position. In practice, solving the modified PB equation in the DPBL formalism yields density maps for water (dipoles) and salt (ions). We illustrate here how these maps can be used to help in the analysis of both low- and high-resolution structural X-ray data. As for the former, we implemented a program, AquaSAXS [2], available as a web server, which uses the solvent density map to represent the hydration layer of a given protein atomic model. This approach improves the model where the hydration layer is defined as a constant layer on the surface. As for the latter, we will present how this approach is suited for electrostatics studies of solvation in confined geometries. In particular, its application to the study of pentameric ligand-gated ion channels helped proposing a plausible ion permeation mechanism [3].[1] Koehl and Delarue (2010) J Chem Phys 132(6) - 064101.[2] Poitevin et al (2011) NAR 39 - W184-9.[3] Sauguet, Poitevin, Murail et al (2013) EMBO J 32(5) - 728-41.

International audience; GLIC is a prokaryotic orthologue of brain pentameric neurotransmitter receptors. Using whole-cell patch-clamp electrophysiology in a host cell line, we show that short-chain di-carboxylate compounds are positive modulators of pHo 5-evoked GLIC activity, with a rank order of action fumarate > succinate > malonate > glutarate. Potentiation by fumarate depends on intracellular pH, mainly as a result of a strong decrease of the pHo 5-evoked current when intracellular pH decreases. The modulating effect of fumarate also depends on extracellular pH, as fumarate is a weak inhibitor at pHo 6 and shows no agonist action at neutral pHo. A mutational analysis of residue-dependency for succinate and fumarate effects, based on two carboxylate-binding pockets previously identified by crystallography (Fourati et al. 2020), shows that positive modulation involves both the inter-subunit pocket, homologous to the neurotransmitter-binding orthotopic site, and the intra-subunit (also called vestibular) pocket. An almost similar pattern of mutational impact is observed for the effect of caffeate, a known negative modulator. We propose, for both di- carboxylate compounds and caffeate, a model where the inter-subunit pocket is the actual binding site, and the region corresponding to the vestibular pocket is required either for inter-subunit binding itself, or for binding-to-gating coupling during the allosteric transitions involved in pore gating modulation.

In all forms of life, DNA polymerases play central roles in genome replication, maintenance and repair. All DNA polymerases have been grouped in different families, using sequence alignments: PolA, PolB, PolC, PolD, PolX, PolY and reverse transcriptases. The only class of DNA polymerases left whose structure and molecular mechanism are unknown is PolD. PolD is an archaeal replicative DNA polymerase made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). To help resolve the uncertainty concerning the evolutionary origins of PolD, I determined the crystal structures of two large fragments of both DP1 and DP2 subunits of the Pyrococcus abyssi PolD. Crystal structures of both DP1 and DP2 subunits revealed that PolD is an atypical DNA polymerase. We also determined the cryo-electron microscopy (cryo-EM) structure of the DP1-DP2 complex bound with DNA. Structures of both polymerase and proofreading active sites differ from other structurally characterized DNA polymerases. In addition, PolD shares an unexpected structural homology with the ‘two-barrel’ family of RNA polymerases. By many aspects, this work provides new insights on the evolutionary history of DNA and RNA polymerases.; Dans toutes les formes de vie, les ADN polymérases jouent un rôle central dans la réplication du génome, sa maintenance et sa réparation. Toutes les ADN polymérases ont été classées en sept familles distinctes en se basant sur leur homologie de séquence : PolA, PolB, PolC, PolD, PolX, PolY et les reverse transcriptases. La dernière famille d’ADN polymérase pour laquelle la structure et le mécanisme moléculaire demeurent inconnus est la PolD. La PolD est l’une des ADN polymérases réplicatives des Archées. C’est une polymérase hétérodimérique composée d’une sous-unité DP1, qui catalyse l’activité de relecture exonucléase 3’-5’, et d’une sous-unité DP2, qui catalyse l’activité de polymérisation 5’-3’. Afin de résoudre l’incertitude concernant les origines évolutives de la PolD, j’ai déterminé la structure cristallographique des domaines catalytiques des sous-unités DP1 et DP2 de Pyrococcus abyssi et la structure du complexe DP1-DP2 lié à l’ADN, par cryo-microscopie électronique (cryo-EM). Ces structures révèlent que la PolD est une ADN polymérase atypique qui diffère de celle des autres ADN polymérases caractérisées à ce jour. De plus, DP2 partage une homologie structurale inattendue avec les ARN polymérases à ‘deux-tonneaux-β’. Ces travaux clarifient les relations évolutives entre toutes les ADN polymérases et mettent en lumière le mécanisme d'acquisition et d'échange de domaines qui s'est produit au cours de l'évolution du réplisome eucaryote.