PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

 

Department: Structural Biology and Chemistry

Title of the PhD project:

Structure, function and regulation of a key metabolic supercomplex in Actinobacteria

 

Name of the lab: Structural Microbiology Unit

Head of the lab: Pedro M. Alzari, Prof.

PhD advisor: Marco Bellinzoni and Pedro M. Alzari

Email address: marco.bellinzoni@pastur.fr; pedro.alzari@pasteur.fr

Web site address of the lab: http://www.pasteur.fr/recherche/unites/Mstruct

Doctoral school affiliation and University: ‘Interdisciplinaire pour le Vivant’ (iViv, ED387), Université ‘Pierre et Marie Curie’

 

 

Presentation of the laboratory and its research topics:

 

The Structural Microbiology Unit at the Institut Pasteur has a long-lasting interest in the structural biology of mycobacteria and has been active member, for more than ten years, of three successive European Commission-funded clusters dedicated to the identification, characterization and validation of new targets for anti-tuberculosis drug development, i.e. the projects ‘X-TB’ (2001-2005), ‘NM4TB’ (2006-2011) and the ongoing project ‘More Medicines for TB’ (‘MM4TB’, 2011-2016), all led by Prof. Stewart T. Cole (EPFL, Lausanne, Switzerland). The activity of the lab has been focused on signal transduction mechanisms ranging from two component systems to Ser/Thr kinases, key signalling elements in Mycobacterium tuberculosis. Our recent work revealed that central metabolism in mycobacteria is controlled by the Ser/Thr kinase PknG (one of the most studied kinases in M. tuberculosis) through GarA, a small FHA-domain protein that regulates the activity of the enzymes situated at the a-ketoglutarate branching point, a crucial crossroad between carbon and nitrogen metabolism.

The project below has recently been selected for funding by the ANR through a ‘Young researcher’ grant to M. Bellinzoni.

 

Description of the project:

 

Tuberculosis remains nowadays a major threat for public health, with approximately one third of the world population estimated to be latently infected with Mycobacterium tuberculosis, according to the WHO [1] and therefore susceptible to develop active disease at anytime through reactivation, especially in case of weakening of the immune response. To become able to target this huge reservoir in an effective way would constitute undoubtedly a great progress towards the eradication of the disease. Indeed, one of the biggest challenges in drug development against tuberculosis is how to hit dormant bacteria, a particularly difficult task in light of the fact that the molecular mechanisms underlying the adaptation of the pathogen to the different environmental conditions during the disease stages, notably dormancy, are still poorly understood. For these reasons we have been actively involved in the study of how M. tuberculosis controls its central metabolism, a critical process in order to switch between active growth and dormancy. Our recent work in this field has been focused on the structural characterization of a-ketoglutarate decarboxylase (KGD), a central enzyme in the tricarboxylic acid cycle (TCA) and part of the tripartite a-ketoglutarate dehydrogenase complex (KDH). First identified as an enzyme inhibited by GarA [2], an FHA-domain protein acting in turn as a sort of molecular switch controlled by phosphorylation, we showed that KGD is an extremely dynamic object able to carry out different reactions depending on the acyl acceptor substrate [3], and that its inhibition by GarA takes place through an unprecedented allosteric mechanism, by which the enzyme is frozen into a catalytically incompetent state [4].

Although the KDH complex was initially reported to be absent in M. tuberculosis due to failure to detect its activity [5], we now know that not only this activity can be measured under appropriate conditions [3], but also that this complex may in reality form a unique ‘supercomplex’ with pyruvate dehydrogenase (PDH), a similarly structured complex involved in the oxidative decarboxylation of pyruvate produced by glycolysis. Indeed, both complexes are known to be huge molecular assemblies structured around a central core, made of either 24 or 60 subunits of the E2 component, and recent data support the existence of such a hybrid supercomplex in Corynebacterium glutamicum [6]. Merging the two complexes would generate a MDa-range multienzyme assembly that could span a diameter larger than the ribosome and without analogies in other organisms. The objective of this project is first to validate the existence of this supercomplex as a physiologically relevant enzymatic machinery in the Corynebacterineae suborder, working on the non-pathogenic species C. glutamicum and M. smegmatis, then to purify and characterize it by a combination of hybrid structural biology approaches, notably cryo-electron microscopy (on the newly installed facilities at the Institut Pasteur), to get a three-dimensional model of this supramolecular biological object. The ultimate goal, however, is to go beyond a static structural picture, clarifying the dynamic processes by which the different enzymatic activities may be temporally and spatially coordinated, and to understand, in the end, by which molecular mechanisms (and in response to which stimuli) such a huge machinery could be regulated.

 

References:

 

  1. WHO. Fact sheet n°104, February 2013. http://www.who.int/mediacentre/factsheets/fs104/en/
  2. O'Hare, H.M., Durán, R., Cerveñansky, C., Bellinzoni, M., Wehenkel, A.M., Pritsch, O., Obal, G., Baumgartner, J., Vialaret, J., Johnsson, K., and Alzari, P.M. (2008) Mol. Microbiol. 70: 1408-1423.
  3. Wagner, T., Bellinzoni, M., Wehenkel. A., O’Hare, H.M. and Alzari, P.M. (2011) Chem. Biol. 18: 1011-1020.
  4. Wagner, T., et al. (2013) Submitted.
  5. Tian, J., Bryk, R., Itoh, M., Suematsu, M., and Nathan, C. (2005) Proc. Natl. Acad. Sci. USA 102: 10670-10675.
  6. Hoffelder, M., Raasch, K., van Ooyen, J, and Eggeling, L. (2010) J. Bacteriol. 192: 5203-5211.

 

Keywords:

 

Mycobacterium, a-ketoglutarate, multi-protein complex, cryo-electron microscopy, structural biology

 

Expected profile of the candidate (optional):

 

The candidate should have a strong interest in structural biology and willing to learn and take advantage of working with multiple techniques (e.g. cryo-electron microscopy, X-ray crystallography, SAXS) and cutting-edge equipment. Some prior experience in any of these methods, as well as in protein purification would be an asset.

 

Contact:

 

Informal enquiries by email are welcome: marco.bellinzoni@pasteur.fr.

 

Mis à jour le 16/09/2013