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.X-ray crystallography | allostery | signal transduction | cys-loop receptor P entameric ligand-gated ion channels (pLGICs) are a superfamily of membrane receptors that mediate fast chemical transmission of nerve signals in the central and peripheral nervous system (1). These allosteric receptors couple neurotransmitter (agonist) binding in the extracellular domain (ECD) to the opening of the ionic pore located in the transmembrane domain (TMD). There are two classes in pLGIC, with either cationic channels (acetylcholine -nAChR -and 5HT3 receptors) or anionic ones (glycine and GABA receptors). The molecular understanding of their allosteric transitions is a central issue in the pharmacology of pLGICs, as it would allow the rational design of novel orthosteric and allosteric ligands. Recently reported full-length structures of several prokaryotic and eukaryotic members of the family have provided significant insights into the conserved architecture of these receptors (2-5). Nevertheless, little is known about the structural events that link the binding/unbinding of agonist to the opening/closure of the channel gate. To understand the gating mechanism, the structures of the same receptor in different allosteric states are needed at atomic resolution. Here we report two crystal structures of wildtype GLIC, a proton-gated bacterial ion channel from Gloeobacter violaceus (6) at two different pHs, above and below pH 50 .GLIC structure wa...

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).

The high-resolution crystal structure of a pentameric ligand-gated ion channel reveals that hydroxylated residues and two water pentagon rings form an ion selectivity filter, explaining ion transport across hydrophobic constriction barriers.

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. The concept of a pharmacological receptor can be traced back more than a hundred years ago to the works of Paul Ehrich and, particularly, John Newport Langley (Langley, 1905), who observed that the neuromuscular junction, whose neurotransmitter is acetylcholine (ACh), was the site of action for nicotine and curare. Langley demonstrated that nicotine behaves as an agonist, causing a contraction of the striated muscles, and that curare, on the other hand, behaves as a competitive antagonist by directly blocking it. The mechanism of signal transduction that results in the opening of a cation selective ion channel (Katz and Miledi, 1966) is a conformational change of the nicotinic acetylcholine receptor (nAChR), the first neurotransmitter receptor and ion channel ever isolated (

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.

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.

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-tRNAPhe 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.

BackgroundDNA 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 FindingsHere we report the crystal structure of the yeast PriL-CTD at 1.55 Å 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/SignificanceOur 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.