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: Deciphering bacterial transmembrane signaling through a heme transporter by NMR
Name of the lab: Unit of NMR of Biomolecules
Head of the lab: Muriel Delepierre
PhD advisor: Nadia Izadi Pruneyre
Email address: firstname.lastname@example.org
Web site address of the lab: http://www.pasteur.fr/recherche/unites/rmnb
Doctoral school affiliation and University: iViv/Complexité du vivant Paris 6
Presentation of the laboratory and its research topics:
We aim at understanding biologically relevant mechanisms at both molecular and atomic scales, through the study of the structure, dynamics and interactions of proteins. To this end, we use NMR, biophysical and biochemical techniques and work in collaboration with various groups and platforms from the Institut Pasteur and with laboratories in France and abroad. Our research is organized around three groups. The candidate will join the group of "bacterial transmembrane signaling" headed by Nadia Izadi Pruneyre.
Description of the project:
Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. Firstly, an external signal is sensed by an outer membrane receptor. Then, a cascade of molecular interactions is triggered to send the signal from the outside to the inside of bacteria. In Gram-negative bacteria, some outer membrane transporters specialized in acquisition of scarce nutrients play such a sensing and signaling function. When a transporter detects the presence of a nutrient, a signal is transmitted, via molecular interactions between proteins located in different subcellular compartments and reaches to the cytoplasmic ECF (extracytoplasmic function) sigma factor which induces the expression of genes related to this nutrient acquisition. In some species, this process also regulates the expression of genes associated with the virulence of the bacteria, allowing a better adaptation to the host environment.
Although the different components of this signaling pathway have been identified, we are still far from understanding the interactions they make during different stages of signal transduction.
The ‘heme acquisition system’ (Has) developed by several gram-negative bacteria to satisfy their need for iron, represents an adequate model to understand this signaling pathway. We have been working for several years on the Has system of Serratia marcescens, the first identified and the most deeply studied. The central protein of the Has system, the outer membrane transporter HasR functions in synergy with an extracellular heme carrier protein, the hemophore HasA (2). HasA either binds free heme or captures it from host hemoproteins (like hemoglobin) and then delivers it to HasR. The presence of heme and hemophore on the extracellular face of the receptor triggers a signal which is then transmitted to two other proteins. One of them, the anti-sigma factor HasS regulates the activity of the ECF-sigma factor. The other, HasB, a TonB-like protein, transduces the energy needed for transport and signaling activity of the transporter. We have shown that the network of interactions between HasB and HasR is modified when the substrates, heme and HasA, are present on the extracellular face of the transporter HasR. The signaling to HasB is based on structural modifications through HasR. We want to determine these structural modifications at the molecular and atomic levels. The structure of HasA, HasR and HasB is known. We use SAXS to analyze the overall structural changes of HasR-HasB complex in the presence or absence of HasA and heme. The aim of this project is to define these structural changes at the atomic level. Presently, the NMR signals of both HasB and its domain of interaction on HasR in free form are completely assigned. All proteins have already been expressed and purified in our laboratory. Although the size of HasR complexes (around 100 kDa embedded in the micelle of detergent) is too large for a NMR study, preliminary experiments show that the domain of interaction of HasR with HasB can be observed and analyzed by this technique. This study can be extended to HasS.
Cardoso de Amorim G, Prochnicka-Chalufour A, Delepelaire P, Lefèvre J, Simenel C, Delepierre M & Izadi-Pruneyre N*.* The structure of HasB reveals a new class of "TonB protein" fold. /PlosOne/2013;8(3):e58964.
Malki I, Cardoso de Amorim G, Simenel C, Prochnicka-Chalufour A, Delepierre M, Izadi-Pruneyre N. (1)H, (13)C and (15)N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter. /Biomol NMR Assign//.
Krieg S, Huché F, Diederichs K, Izadi-Pruneyre N, Lecroisey A, Wandersman C, Delepelaire P, Welte W. Heme uptake across the outer membrane as revealed by crystal structures of the receptor-hemophore complex. /Proc Natl Acad Sci U S A/. 2009 Jan 27;106(4):1045-50
Caillet-Saguy C, Piccioli M, Turano P, Izadi-Pruneyre N, Delepierre M, Bertini I, Lecroisey A. Mapping the interaction between the hemophore HasA and its outer membrane receptor HasR using CRINEPT-TROSY NMR spectroscopy. /J Am Chem Soc//./ 2009 Feb 11;131(5):1736-44.
Lefèvre J, Delepelaire P, Delepierre M, Izadi-Pruneyre N. Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB. /J Mol Biol/. 2008 May 9;378(4):840-51
Comparative analysis of structural and dynamic properties of the loaded and unloaded hemophore HasA: functional implications. Wolff N, Izadi-Pruneyre N, Couprie J, Habeck M, Linge J, Rieping W, Wandersman C, Nilges M, Delepierre M, Lecroisey A. J Mol Biol. 2008.
Structural Biology, Membrane protein complex, signaling, iron acquisition by bacteria
Expected profile of the candidate (optional):
A background in biochemistry and purification and characterization of protein is required. Experience in NMR is useful but not necessary.