Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate. Corrigendum

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

Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors.Fil: Trajtenberg, Felipe. Instituto Pasteur de Montevideo; UruguayFil: Albanesi, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Ruetalo, Natalia. Instituto Pasteur de Montevideo; UruguayFil: Botti, Horacio. Instituto Pasteur de Montevideo; UruguayFil: Mechaly, Ariel. Instituto Pasteur de Montevideo; Uruguay. Institut Pasteur, Unité de Microbiologie Structurale, Paris; FranciaFil: Nieves, Marcos. Instituto Pasteur de Montevideo, Laboratorio de Biología Celular de Membranas, Montevideo; UruguayFil: Aguilar, Pablo Sebastián. Instituto Pasteur de Montevideo, Laboratorio de Biología Celular de Membranas, Montevideo; UruguayFil: Cybulski, Larisa Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Larrieux, Nicole. Instituto Pasteur de Montevideo; UruguayFil: de Mendoza, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Buschiazzo, Alejandro. Instituto Pasteur de Montevideo; Uruguay. Institut Pasteur, Département de Biologie Structurale et Chimie, Paris; Franci

Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography

International audienceWe have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR complexes and further purification. The 3D structures of the complex trapped in the phosphotransferase and in the phosphatase reaction steps, were solved by X-ray crystallography using molecular replacement. The solution was not trivial, and we found that in silico-generated models used as search probes, were instrumental to succeeding in placing a large portion of the complex in the asymmetric unit. Electron density maps were then clear enough to allow for manual model building attaining complete atomic models. These methods contribute to tackling a major challenge in the bacterial signaling field, namely obtaining stable kinase:regulator complexes, in distinct conformational states, amenable for high-resolution crystallographic studies

Structural insights into bacterial resistance to cerulenin

International audienceCerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin

Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis

International audienceThe protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. 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 pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA from L. interrogans was 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 species L. 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

Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action

International audienceTwo-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

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization

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Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine

International audienceMycobacterium 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

Reactivity of mAb 13G9 with <i>T. cruzi</i> parasites.

<p><b>A</b>) <i>T. cruzi</i> surface labeling by the 13G9 mAb. Epifluorescence microscopy of <i>T. cruzi</i> trypomastigotes, seeded onto poly-L-lysine-treated coverglasses, and immunolabeled with 13G9 mAb followed by a secondary FITC-labeled antibody. <b>B</b>) Inhibition of parasite sialylation. Trypomastigotes obtained from cell cultures made in ‘low sialyl-donors’ conditions, were sialylated with TS and sialyllactose, in the presence of mAb 13G9. Total sialic acid was quantified by the thiobarbituric acid method and referred to re-sialylated parasites in the absence of mAb as 100% (approximately 1.2 pmoles of sialic acid/10⁶ parasites). <b>C</b>) Effect of mAb 13G9 in infection assays on mammalian cells. Parasites were preincubated for 1 h with 13G9 antibody (100 µg/ml) before infection. After 24 hrs, infected cultures were fixed and stained with Hoescht 33342. At least 300 cells were counted.</p