Structure function analysis of pseudopilus assembly and type II protein secretion

A two-year post-doctoral position funded by the ANR (The French National Research Agency) grant is available from January 2015 in the team of Olivera Francetic, Laboratory of Macromolecular Systems and Signaling in the Institute Pasteur in Paris, France.

 


The host laboratory is interested in understanding the structural and mechanistic basis of protein transport and fiber assembly by the type II secretion systems (T2SS).  Reconstituted in Escherichia coli, the T2SS from Klebsiella oxytoca allows transport of a folded substrate protein (pullulanase) from the periplasm to the cell surface. An essential step in the secretion process is the assembly of a periplasmic fiber, the pseudopilus, whose elongation drives protein transport via as yet unknown mechanism. Pseudopili are assembled in the inner membrane base from five pseudopilin subunits. The major subunit PulG forms long homopolymers that can extend artificially beyond the cell surface. Detailed structure function analysis of this homopolymer allowed us to identify its assembly and stability determinants and to propose a rotational fiber assembly mechanism (1, 2). Other studies identified the role of three of the minor pseudopilins, PulI, PulJ and PulK in the initiation step of pseudopilus assembly (3). The fourth minor pseudopilin PulH ensures an efficient transition towards the elongation phase and towards the assembly of PulG homopolymers. This phase is catalyzed by the PulE cytoplasmic ATPase, which transduces the conformational changes to the assembly complex in the inner membrane. Several questions are currently open: how does PulH couple assembly initiation to fiber elongation?  How does the ATPase PulE transfer the energy to the assembly complex to polymerize pseudopili? How is the pseudopilus assembly coupled to protein secretion? To address these questions the successful candidate will have at his/her disposal numerous genetic and biochemical tools generated in the lab as well as the state-of-the-art facilities in a highly interactive environment of the Institut Pasteur in Paris. The work will be done in close collaboration with specialists in protein structural and interaction analysis by NMR, structural and molecular modeling and  high-resolution electron microscopy.

 

We are seeking highly motivated candidates interested in the mechanistic understanding of biological nanomachines and in function of dynamic protein complexes. Applicants should have a strong expertise in molecular biology and protein biochemistry. Interest and experience in the study of protein structure, protein transport, or membrane protein biogenesis is a plus.

 


Interested candidates should send a Curriculum vitae, a letter of motivation and names and contact information of at least two referees to Olivera Francetic at ofrancet@pasteur.fr.

 

References:

 

1. Campos M, Nilges M, Cisneros DA and O. Francetic (2010) Detailed structural and assembly model of the type II secretion pilus from sparse data. Proc. Natl. Acad. Sci. USA 107: 13081-86.

 

2. Nivaskumar, M, Bouvier, G, Campos, M, Yu, X, Nadeau, N, Egelman, E, Nilges, M and Francetic O. (2014) Distinct docking and stabilization steps of the pseudopilus transition path suggest rotational assembly of type IV pilus-like fibers. Structure. 2014 22(5): 685-96.

 

3. Cisneros, DA, Bond PJ, Pugsley AP, Campos M and O. Francetic. (2012) Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation. EMBO J. 31: 1041-1053.

Mis à jour le 03/09/2014

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