|
|
 |
|
Heavy metal homeostasisGroup leader : Jacques Covès Group
IntroductionCupriavidus metallidurans CH34 is the archetype of heavy metal resistant bacteria. As its genome is now fully sequenced (http://genome.ornl.gov/microbial/rmet/), this strain is an ideal organism both for bioremediation processes and for studying the basic mechanisms of heavy metal homeostasis. Certain metals are essential oligo-elements for all living organisms and deficiency or excess can be deleterious. Others are simply toxic. Their intracellular concentration must be highly regulated. This control is mediated by the inducible expression of specific genes, by transport proteins mediating the traffic of the metal through the membranes, by small proteins delivering the metal to specific targets, the metallo-chaperones. The group has two main topics:
Role of the periplasm in the resistance to copper (J. Covès)Copper is a vital mineral nutrient required for the growth and development of living organisms, including bacteria. It is a transition metal essential for the activity of a wide variety of enzymes. However, this metal poses a dilemma for living systems. The same redox chemistry that makes copper essential also makes it a potent toxic compound when the homeostatic balance controlling the level of free copper ions fails. Free copper ions participate in redox reactions that generate free radicals or oxidative damage. These conflicting properties demand close regulation of copper level. Virtually all cells have developed sophisticated homeostasis mechanisms to tightly control copper uptake and its mobilization to appropriate target proteins and compartments. However, significant gaps remain in our understanding of copper metabolism. At a cellular level, basic components of copper uptake, intracellular transport, export and traffic remain undefined as well as the dynamic regulation of the homeostasis mechanisms. At a molecular level, a fine characterization of the different copper-binding proteins involved in homeostasis and resistance is still missing. This characterization includes the structure, the metal-binding properties, the putative enzymatic activities (copper reductase, copper oxidase) and also the possibility for these proteins to transiently interact in a network. The question of how copper is handled in the periplasmic space of Gram-negative bacteria remains open. This question is of importance because it has been observed that the mechanism of copper resistance is heavily dependent on copper sequestration and accumulation in the periplasm and the outer membrane. The periplasm contains also sensing proteins able to detect subtle variations in heavy-metal external concentrations and to forward the information to cytoplasmic partner proteins (see Topic 2). We are presently involved in the characterization of a series of novel periplasm-located proteins whose exact role and structure are still unknown. Our efforts are already illustrated by the over-expression, the purification and, the biochemical and biophysical characterization of 3 of these proteins. CopK:Transcriptomic and proteomic data showed that this protein is synthesized in high amounts in the presence of copper. Significant sequence similarity could only be found with orthologs of unknown function. We have determined the three-dimensional structure of CopK using NMR in collaboration with Beate Bersch and coworkers. The protein consists of two ß-sheets in a perpendicular orientation and a mobile C-terminal of ten residues. Different experimental NMR parameters indicate that the second ß-sheet is more flexible than the first one. Mass spectrometry and NMR data showed that CopK selectively binds up to two Cu(I) atoms and that metal-binding leads to a slight increase in protein flexibility [1].
The solution structure of CopK has been solved CopH: It is a dimer able to bind 2 Cu(II) ions with high specificity [2]. The spectral characteristics of Cu-bound CopH are in agreement with type 2 Cu(II) in a nitrogen ligand field. We have strong evidence that the two histidine residues are not sufficient to provide this nitrogen ligand field.
Spectrophysical characterization of CopH demonstrates the existence of one Cu(II) type 2 site per monomer CzcE: belongs to the cobalt, zinc and cadmium resistance determinant but we have discovered that it is actually a copper-binding protein. We suggest that CzcE could be an alarm protein sensing copper in case of multi-elemental pollutions. CzcE can exist under 4 redox states: apo-CzcE, oxidized CzcE (4 Cu2+), reduced CzcE (4 Cu1+), semi-reduced CzcE (2 Cu2+ and 2 Cu1+). The spectral signatures of these different redox states reveal ligand changes and protein movements [3]. Apo-CzcE has already been crystallized [4].
[1] Bersch B., Favier A., Schanda P., van Aelst S., Vallaeys T., Covès J., Mergeay M. and Wattiez R.(2008) Molecular structure and metal-binding properties of the periplasmic CopK protein involved in copper detoxification in Cupriavidus metallidurans CH34 J. Mol. Biol 380, 386-403 [2] Cannella D., Bersch B., Fieschi F., Ménage S., Lascoux D. and Covès J. (2006) CopH from Cupriavidus metallidurans CH34. A novel periplasmic copper-binding protein Biochemistry 47, 5557-5566. [3] Zoropogui A., Gambarelli S. and Covès J. (2008) CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein Biochem. Biophys. Res. Commun., 365, 735-739. [4] Pompidor G., Zoropogui A., Kahn R. and Covès J. (2007) Overproduction, purification and preliminary X-ray diffraction analysis of CzcE from Cupriavidus metallidurans CH34 Acta Cryst F63, 884-886. Characterization of signal transduction systems operating from the cytoplasmic membraneLiving cells survive stress factors that they meet in their environment because they build an appropriate response to deal with them. Detection systems of the ECF-type [Extra-Cytoplasmic Function] are used by bacteria to initiate responses to stress factors targeting their protective envelope. In the simplest form, an ECF-type transduction system is made of three polypeptides that work together to make a meaningful signal for the expression machinery out of the stress that the bacterial cell needs to cope with. Cupriavidus metallidurans CH34 uses ECF-type transduction systems to induce its resistance to various metals. Similarly, ECF-type systems contribute to the virulence of several bacterial pathogens, allowing them to induce what could be considered as a response to aggressions from the host which they parasite. As a consequence, medical, environmental and biotechnological benefits are expected from the understanding of structure:function relationships of ECF-type transduction systems. The project ‘Cnr’ aims at characterizing the interactions between the 3 proteins of the CnrYXH signal transduction system of C. metallidurans CH34. CnrYXH regulates the expression of the cnrCBA operon, whose products are responsible for cobalt and nickel resistance of C. metallidurans CH34. This is a biochemistry project based on published genetics and microbiology studies, and which will develop at the interplay of an array of biophysical technics for which expertise is available at the IBS. The role of each of the 3 proteins CnrX, CnrY and CnrH has been established on the basis of their sequences and on the results of genetic studies in the original bug: C. metallidurans CH34. CnrX is a periplasmic sensor of metal. In this respect, CnrX will be studied very much like metal-binding proteins from topic 1. CnrY is a transmembrane protein and interacts both with CnrX on the periplasmic side and with CnrH on the cytoplasmic side. Finally, CnrH is a sigma factor, i.e. a small exchangeable subunit of the bacterial RNA polymerase: CnrH allows the RNA polymerase to specifically bind cnrp promoters, which control the expression of the operons cnrYXH and cnrCBA. Thus CnrH is essential to the transcription of the resistance genes cnrCBA.
Left : at low cobalt and nickel concentrations, the system is at rest. CnrH is sequestered at the membrane by CnrY, which is therefore named “anti-sigma” factor. CnrH is then unavailable for the RNA polymerase and the genes controled by cnrp are not transcribed. The system remains in the resting state until the periplasmic concentration of the metal reaches a threshold. Right : the binding of the metal to the histidine-rich domain of CnrX would induce a conformational change in CnrX that would also impact CnrY conformation. As a consequence of these conformational changes, CnrH is released in the cytoplasm where it is recruited by the RNA polymerase. With CnrH as its sigma factor, the RNA polymerase binds to cnrp promoters. The genes cnrCBA are then transcribed and the efflux pump CnrCBA will be built, thus enhancing C. metallidurans resistance to cobalt and nickel. Main collaborations
Technical approachesBiochemical and metal-binding characterization of the proteins of interest uses the resources of molecular biology, protein biochemistry as well as those of biophysical techniques required for the study of proteins in general and metalloproteins in particular (EPR, X-ray, NMR, CD, Mass Spec, Surface Plasmon Resonance, ITC, XAS [Exafs, Xanes]). The characterization of protein/protein interactions uses chomatography, native electrophoresis, pull-down, covalent cross-linking. All these techniques are available in the IBS or in collaboration with our local partners such as the PSB/CISB or the IMBG (Institut des Métaux en Biologie de Grenoble). The existence of the IMBG ensures the local availability of most techniques as well as the technical know-how related to the characterization of metalloproteins by different spectroscopies. Some recent and relevant publications of the groupZoropogui A., Gambarelli S. and Covès J. (2008) CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein Biochem. Biophys. Res. Commun.365, 735-739. Maillard A.P., Lalani S., Silva F., Belin D. and Duong F. (2007) Deregulation of the SecYEG translocation channel upon removal of the plug domain. J. Biol. Chem. 282, 1281-1287 Stroebel D., Sendra V., Cannella D., Helbig K., Nies D.H. and Covès J. (2007) Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent. Biochim. Biophys. Acta 1768,1567-1573 Sendra V., Cannella D., Bersch B., Fieschi F., Ménage S., Lascoux D. and Covès J. (2006) CopH from Cupriavidus metallidurans CH34. A novel periplasmic copper-binding protein Biochemistry 47, 5557-5566. Avoscan L., Collins R., Carrière M., Gouget B. and Covès J. (2006) Seleno-L-methionine is the predominant organic form of selenium in Cupriavidus metallidurans CH34 exposed to selenite or selenate. Appl. Environ. Microbiol. 72, 6414-6416. Ledgham F., Quest B., Vallaeys T., Mergeay M. and Covès J. (2005) A probable link between the DedA protein and resistance to selenite. Res. Microbiol.156, 367-374. Tam P.C., Maillard A.P., Chan K.K. and Duong F. (2005) Investigating the SecY plug movement at the SecYEG translocation channel. EMBO J. 24, 3380-3388. Maillard A.P., Biarrotte-Sorin S.,Villet R., Mesnage S., Bouhss A., Sougakoff W., Mayer C. and Arthur M. (2005) Structure-based site-directed mutagenesis of the UDP-MurNAc- pentapeptide-binding cavity of the FemX alanyl transferase from Weissella viridescens. J. Bacteriol. 187, 3833-3838 Champier L., Duarte V., Michaud-Soret I. and Covès J. (2004) Characterization of the MerD protein from Ralstonia metallidurans CH34. A possible role in bacterial mercury resistance by switching off the induction of the mer operon Mol. Microbiol., 52, 1475-1485. |
