An original potentiating mechanism revealed by the cryo-EM structures of the human α7 nicotinic receptor in complex with nanobodies.

The human α7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest in designing ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of α7 ligands, we recently generated two nanobodies named E3 and C4, acting as positive allosteric modulator and silent allosteric ligand, respectively. Here, we solved the cryo-electron microscopy structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an agonist-bound conformation of the extracellular domain in the absence of an orthosteric agonist, and mutational analysis shows a key contribution of an N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation that opens new avenues for drug design.

Generation of nanobodies acting as silent and positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR), a potential drug target for treating cognitive disorders, mediates communication between neuronal and non-neuronal cells. Although many competitive antagonists, agonists, and partial-agonists have been found and synthesized, they have not led to effective therapeutic treatments. In this context, small molecules acting as positive allosteric modulators binding outside the orthosteric, acetylcholine, site have attracted considerable interest. Two single-domain antibody fragments, C4 and E3, against the extracellular domain of the human α7-nAChR were generated through alpaca immunization with cells expressing a human α7-nAChR/mouse 5-HT3A chimera, and are herein described. They bind to the α7-nAChR but not to the other major nAChR subtypes, α4ß2 and α3ß4. E3 acts as a slowly associating positive allosteric modulator, strongly potentiating the acetylcholine-elicited currents, while not precluding the desensitization of the receptor. An E3-E3 bivalent construct shows similar potentiating properties but displays very slow dissociation kinetics conferring quasi-irreversible properties. Whereas, C4 does not alter the receptor function, but fully inhibits the E3-evoked potentiation, showing it is a silent allosteric modulator competing with E3 binding. Both nanobodies do not compete with α-bungarotoxin, localizing at an allosteric extracellular binding site away from the orthosteric site. The functional differences of each nanobody, as well as the alteration of functional properties through nanobody modifications indicate the importance of this extracellular site. The nanobodies will be useful for pharmacological and structural investigations; moreover, they, along with the extracellular site, have a direct potential for clinical applications.

A Tetratricopeptide Repeat Scaffold Couples Signal Detection to OdhI Phosphorylation in Metabolic Control by the Protein Kinase PknG.

Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of posttranslational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here, we present an investigation using in vivo experiments, as well as biochemical and structural methods, of the molecular basis of the regulation of PknG from Corynebacterium glutamicum (CgPknG), in the light of previous knowledge available for the kinase from Mycobacterium tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPRs) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain to a ß-hairpin within the flexible N-terminal region that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCE Bacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. Actinobacteria, one of the largest bacterial phyla of major importance for biotechnology, medicine, and agriculture, developed a unique control process that revolves around a key protein, the protein kinase PknG. Here, we use genetic, biochemical, and structural approaches to study PknG in a system that regulates glutamate production in Corynebacterium glutamicum, a species used for the industrial production of amino acids. The reported findings are conserved in related Actinobacteria, with broader significance for microorganisms that cause disease, as well as environmental species used industrially to produce amino acids and antibiotics every year.

Concatemers to re-investigate the role of α5 in α4ß2 nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, ß2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4ß2 and α4ß2α5 receptors and characterized their properties by two-electrodes voltage-clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and ß2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.

The desensitization pathway of GABAA receptors, one subunit at a time.

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, the conformational pathway of which remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1ß2γ2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the γ2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.

Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling.

Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA-binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.

The crystal structure of PknI from Mycobacterium tuberculosis shows an inactive, pseudokinase-like conformation.

Eukaryotic-like Ser/Thr protein kinases (ePKs) have been identified in many bacterial species, where they are known to mediate signalling mechanisms that share several features with their eukaryotic counterparts. In Mycobacterium tuberculosis, PknI is one of the 11 predicted ePKs and it has been related to bacterial virulence. In order to better understand the molecular basis of its role in mycobacterial signalling, we solved the crystal structure of the PknI cytoplasmic domain. We found that even though PknI possesses most conserved elements characteristic of Hanks-type kinases, it is degraded in several motifs that are essential for the ePKs catalytic activity. Most notably, PknI presents a remarkably short activation segment lacking a peptide-substrate binding site. Consistent with this observation and similar to earlier findings for eukaryotic pseudokinases, no kinase activity was detected for the catalytic domain of PknI, against different substrates and in various experimental conditions. Based on these results, we conclude that PknI may rely on unconventional mechanism(s) for kinase activity and/or it could play alternative role(s) in mycobacterial signalling. DATABASE: Atomic coordinates and structure factors for the catalytic domain of M. tuberculosis PknI are in the Protein Data Bank under the accession codes 5M06 (wild-type PknI + ADP), 5M07 (PknI_C20A), 5M08 (PknI_C20A_R136A) and 5M09 (PknI_C20A_R136N).

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

Molecular Basis of the Activity and the Regulation of the Eukaryotic-like S/T Protein Kinase PknG from Mycobacterium tuberculosis.

Tuberculosis remains one of the world's deadliest human diseases, with a high prevalence of antibiotic-resistant Mycobacterium tuberculosis (Mtb) strains. A molecular understanding of processes underlying regulation and adaptation of bacterial physiology may provide novel avenues for the development of antibiotics with unconventional modes of action. Here, we focus on the multidomain S/T protein kinase PknG, a soluble enzyme that controls central metabolism in Actinobacteria and has been linked to Mtb infectivity. Our biochemical and structural studies reveal how different motifs and domains flanking the catalytic core regulate substrate selectivity without significantly affecting the intrinsic kinase activity, whereas a rubredoxin-like domain is shown to downregulate catalysis through specific intramolecular interactions that modulate access to a profound substrate-binding site. Our findings provide the basis for the selective and specific inhibition of PknG, and open new questions about regulation of related bacterial and eukaryotic protein kinases.

The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation.

Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases.

Secondary structure reshuffling modulates glycosyltransferase function at the membrane.

Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including ß-strand-to-α-helix and α-helix-to-ß-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.

A dual conformation of the post-decarboxylation intermediate is associated with distinct enzyme states in mycobacterial KGD (α-ketoglutarate decarboxylase).

α-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of α-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by α-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain α-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the α-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (α-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o.

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis.

Alpha-ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α-ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non-pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient-dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.

Conformational changes upon ligand binding in the essential class II fumarase Rv1098c from Mycobacterium tuberculosis.

rv1098c, an essential gene in Mycobacterium tuberculosis, codes for a class II fumarase. We describe here the crystal structure of Rv1098c in complex with l-malate, fumarate or the competitive inhibitor meso-tartrate. The models reveal that substrate binding promotes the closure of the active site through conformational changes involving the catalytic SS-loop and the C-terminal domain, which likely represents a general feature of this enzyme superfamily. Analysis of ligand-enzyme interactions as well as site-directed mutagenesis suggest Ser318 as one of the two acid-base catalysts.

Biosynthetic origin of the galactosamine substituent of Arabinogalactan in Mycobacterium tuberculosis.

The arabinogalactan (AG) of slow growing pathogenic Mycobacterium spp. is characterized by the presence of galactosamine (GalN) modifying some of the interior branched arabinosyl residues. The biosynthetic origin of this substituent and its role(s) in the physiology and/or pathogenicity of mycobacteria are not known. We report on the discovery of a polyprenyl-phospho-N-acetylgalactosaminyl synthase (PpgS) and the glycosyltransferase Rv3779 from Mycobacterium tuberculosis required, respectively, for providing and transferring the GalN substrate for the modification of AG. Disruption of either ppgS (Rv3631) or Rv3779 totally abolished the synthesis of the GalN substituent of AG in M. tuberculosis H37Rv. Conversely, expression of ppgS in Mycobacterium smegmatis conferred upon this species otherwise devoid of ppgS ortholog and any detectable polyprenyl-phospho-N-acetylgalactosaminyl synthase activity the ability to synthesize polyprenyl-phospho-N-acetylgalactosamine (polyprenyl-P-GalNAc) from polyprenyl-P and UDP-GalNAc. Interestingly, this catalytic activity was increased 40-50-fold by co-expressing Rv3632, the encoding gene of a small membrane protein apparently co-transcribed with ppgS in M. tuberculosis H37Rv. The discovery of this novel lipid-linked sugar donor and the involvement of a the glycosyltransferase C-type glycosyltransferase in its transfer onto its final acceptor suggest that pathogenic mycobacteria modify AG on the periplasmic side of the plasma membrane. The availability of a ppgS knock-out mutant of M. tuberculosis provides unique opportunities to investigate the physiological function of the GalN substituent and the potential impact it may have on host-pathogen interactions.

AftD, a novel essential arabinofuranosyltransferase from mycobacteria.

Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.

Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice.

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the alpha-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two alpha-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.

Synthesis and biochemical evaluation of selective inhibitors of class II fructose bisphosphate aldolases: towards new synthetic antibiotics.

We report the synthesis and biochemical evaluation of selective inhibitors of class II (zinc-dependent) fructose bisphosphate aldolases. The most active compound is a simplified analogue of fructose bisphosphate, bearing a well-positioned metal chelating group. It is a powerful and highly selective competitive inhibitor of isolated class II aldolases. We report crystallographic studies of this inhibitor bound in the active site of the Helicobacter pylori enzyme. The compound also shows activity against Mycobacterium tuberculosis isolates.

A point mutation in the two-component regulator PhoP-PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra.

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.

Isoxyl activation is required for bacteriostatic activity against Mycobacterium tuberculosis.

Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoyl-coenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis.