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Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control., FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis in Mycobacterium tuberculosis. Here authors present crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms.

Protein Kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. The activation of the holoenzyme depends on the binding of the second messenger cAMP. The only available structures of the D/D domain proceed from mammalian sources. Unlike dimeric mammalian counterparts, the R subunit from Saccharomyces cerevisiae (Bcy1) forms tetramers in solution. Here we describe the first high-resolution structure of a non-mammalian D/D domain. The tetramer in the crystals of the Bcy1 D/D domain is a dimer of dimers that retain the classical D/D domain fold. By using phylogenetic and structural analyses combined with site-directed mutagenesis, we found that fungal R subunits present an insertion of a single amino acid at the D/D domain that shifts the position of a downstream, conserved arginine. This residue participates in intra-dimer interactions in mammalian D/D domains, while due to this insertion it is involved in inter-dimer contacts in Bcy1, which are crucial for the stability of the tetramer. This surprising finding challenges well-established concepts regarding the oligomeric state within the PKAR protein family and provides important insights into the yet unexplored structural diversity of the D/D domains and the molecular determinants of R subunit oligomerization.

Phytoplasmas are wall-less phytopathogenic bacteria that produce devastating effects in a wide variety of plants. Reductive evolution has shaped their genome, with the loss of many genes, limiting their metabolic capacities. Owing to the high concentration of C4 compounds in plants, and the presence of malic enzyme (ME) in all phytoplasma genomes so far sequenced, the oxidative decarboxylation of l-malate might represent an adaptation to generate energy. Aster yellows witches’-broom (Candidatus Phytoplasma) ME (AYWB-ME) is one of the smallest of all characterized MEs, yet retains full enzymatic activity. Here, the crystal structure of AYWB-ME is reported, revealing a unique fold that differs from those of ‘canonical’ MEs. AYWB-ME is organized as a dimeric species formed by intertwining of the N-terminal domains of the protomers. As a consequence of such structural differences, key catalytic residues such as Tyr36 are positioned in the active site of each protomer but are provided by the other protomer of the dimer. A Tyr36Ala mutation abolishes the catalytic activity, indicating the key importance of this residue in the catalytic process but not in the dimeric assembly. Phylogenetic analyses suggest that larger MEs (largesubunit or chimeric MEs) might have evolved from this type of smaller scaffold by gaining small sequence cassettes or an entire functional domain. The Candidatus Phytoplasma AYWB-ME structure showcases a novel minimal structure design comprising a fully functional active site, making this enzyme an attractive starting point for rational genetic design. Fil: Alvarez, Clarisa Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Trajtenberg, F.. Instituto Pasteur de Montevideo; Uruguay Fil: Larrieux, N.. Instituto Pasteur de Montevideo; Uruguay Fil: Saigo, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Golic, Adrián Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Andreo, Carlos Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Hogenhout, S. A.. John Innes Institute; Reino Unido Fil: Mussi, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Drincovich, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Buschiazzo, A.. Instituto Pasteur de Montevideo; Uruguay

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genusLeptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenicLeptospiraparticularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA fromL. interroganswas purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic speciesL. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

Two-component systems (TCS) are protein machineries that enable cells to respond to input signals. Histidine kinases (HK) are the sensory component, transferring information toward downstream response regulators (RR). HKs transfer phosphoryl groups to their specific RRs, but also dephosphorylate them, overall ensuring proper signaling. The mechanisms by which HKs discriminate between such disparate directions, are yet unknown. We now disclose crystal structures of the HK:RR complex DesK:DesR from Bacillus subtilis, comprising snapshots of the phosphotransfer and the dephosphorylation reactions. The HK dictates the reactional outcome through conformational rearrangements that include the reactive histidine. The phosphotransfer center is asymmetric, poised for dissociative nucleophilic substitution. The structural bases of HK phosphatase/phosphotransferase control are uncovered, and the unexpected discovery of a dissociative reactional center, sheds light on the evolution of TCS phosphotransfer reversibility. Our findings should be applicable to a broad range of signaling systems and instrumental in synthetic TCS rewiring. DOI: http://dx.doi.org/10.7554/eLife.21422.001

International audience; An N-terminally truncated version of the enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi lacking the first 37 residues was crystallized both in its apo form and in a binary complex with glucose 6-phosphate. The crystals both belonged to space group P2(1) and diffracted to 2.85 and 3.35 Å resolution, respectively. Self-rotation function maps were consistent with point group 222. The structure was solved by molecular replacement, confirming a tetrameric quaternary structure.

Two-component systems (TCSs) are key players in bacterial signaling, to better understand signal-transmission with molecular detail. The TCS DesK/DesR controls fatty acid desaturation in Bacillus subtilis in response to cold shock and other membrane-altering effectors. We had previously put forward a model of signal-dependent allosteric control of the sensor kinase catalytic activity [1,2]. We have now turned our attention to the response regulator DesR. A canonical activation pathway has been widely accepted to explain phosphorylation-mediated control of response regulator function, allosterically coupling the phosphorylation site to the α4β5α5 surface. However, the structural evidence supporting the main hypotheses is still highly fragmentary. We are now reporting the crystal structure of full-length DesR, in complex with a phosphoryl-mimetic, showing the activated state [3]. Several crystal forms of the receiver domain were determined in the active and inactive configurations, revealing molecular details of the activation switch. Comparative small angle X ray scattering of full-length constructs, structure-guided point mutagenesis, as well as in vitro and in vivo functional analyses, allow us to propose an integral model of DesR activation. The phosphorylation of the receiver domain is allosterically coupled not to one, but two exposed surfaces, independently controlling its dimerization and tetramerization. Notably, a novel surface is shown to be essential for a non-canonical dimerization and activation mechanism. Direct coupling analysis highlights this interface as a shared feature of all NarL/LuxR regulators. This surface is further involved in cognate histidine kinase binding, disclosing a novel view of response regulator allosteric control. With the data we are now reporting, the DesK/DesR signaling pathway becomes, to the best of our knowledge, one of the most thoroughly studied examples of a thermosensor TCS at the molecular and biological levels.

A correction to the article by Ortíz et al. [(2011), Acta Cryst. F67, 1457–1461]., A figure in the article by Ortíz et al. [(2011), Acta Cryst. F67, 1457–1461] is corrected.