PhD projects

ITP FP7 - SBMPs

Structural Biology of Membrane Proteins

PhD project:

Structural study of the E. coli-bacteriophage T5 recognition mechanisms

A combined electron microscopy and crystallographic approach




Principal Investigator:

C. Breyton, J.-L. Popot, C.N.R.S./Université Paris-7 UMR 7099, Institut de Biologie Physico-Chimique, 13, rue Pierre-et-Marie-Curie, F-75005 Paris, France; Cecile.Breyton|ibpc.fr, Jean-Luc.Popot|ibpc.fr


In collaboration with:

M. Chami, A. Engel, M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, 4056 Basel, Switzerland; Mohamed.Chami|unibas.ch, Andreas.Engel|unibas.ch

P. Boulanger, CNRS UMR 8619, IBBMC, Université de Paris-Sud, Bâtiment 430, F-91405 ORSAY Cedex, France; Pascale.Boulanger-Biard|u-psud.fr

Infection of Gram-negative bacteria by a bacteriophage is initiated by the irreversible binding of the phage to an outer membrane receptor of the host.  In the case of tailed phages, this interaction triggers conformational changes that are transmitted from the tail tip to the capsid, allowing its opening and the release of the viral genome.  The latter is then transferred via the tail, through the host envelope, into the cytoplasm of the bacterium. We are interested in these early infection steps at the molecular and structural level in the case of the E. coli-phage T5 system, for which the different actors are known:

i) the outer membrane iron-ferrichrome transporter FhuA is phage T5’s receptor located at the surface of E. coli. FhuA is a monomeric β-barrel comprised of 22 transmembrane strands. The lumen of the barrel is closed by the N-terminal domain, which forms a plug inside it1, 2.

ii) the receptor binding protein (RBP) pb5 (68.5 kDa), located at the tip of the tail of phage T5, has been cloned, over-expressed and purified.  The 1:1 complex it forms with FhuA in vitro is highly stable, as it is not denatured by 2% SDS up to 70°C3. pb5 binding to FhuA initiates the infection process, probably through conformational changes that are transmitted along the tail to the capsid.  These events lead to the penetration of the straight fiber, mainly constituted by the protein pb2, thus forming a channel through the whole bacterial envelop, allowing the DNA into the bacterium.

iii) pb2 is a multidomain, multimeric protein of 124 kDa.  Domain I, predicted to be strongly coiled-coiled is believed to be the tail’s “tape measure protein”, and thought to serve as a sensor for triggering the opening of the capsid.  Domain II contains two hydrophobic α-helices, and domain III a peptidoglycane hydrolysis activity.  A truncated version of pb2, containing the last two domains, has been overexpressed and characterised.  Purified pb2-Cterm behaves as a transmembrane oligomeric protein with fusogenic activity4, 5. So far, full-length over-expressed pb2 could not be correctly refolded, presumably because of its high content of coiled-coil domains.


The stoichiometric complex formed by FhuA and pb5 is one of the very few available receptor/RBP complexes.  We are interested in its structure to help understanding how the binding of the phage to the bacterium induces phage infection.  The structure of pb5 alone will also be investigated, for pb5 conformational changes are most probably the key to the communication with the rest of the phage tail for DNA release.  The complex will be studied by single-particle imaging in negative stain and by 2D electron crystallography to obtain a low-resolution envelope.  3D crystallisation, to obtain atomic resolution information, will be performed both on the complex and on pb5 alone.


The structure of the membrane protein pb2-Cterm will be investigated by both 2D and 3D crystallisation.  Different constructions of pb2 will be studied.  Indeed, it has been shown that pb2 undergoes specific proteolytic cleavage following phage interaction with FhuA and DNA release.  Identification of the proteolytic fragments could provide interesting candidates for structural studies.


We are also interested in the reconstitution of the phage straight fiber, i.e. the interaction between pb5 and pb2. pb5 and different constructions of pb2 will thus be co-expressed and conditions optimised to favour their interaction. pb3 and pb4, two hypothetical base plate proteins, might need to be included to promote formation of the straight fiber. The resulting architecture will be investigated by single particle electron microscopy.


- 3D crystallisation of the different partners (C. Breyton/P. Boulanger, 24 months)

Over-expression, purification, characterisation of FhuA, pb5 and pb2-Cterm will be carried out in our labs. Small crystals of the FhuA-pb5 complex have recently been obtained. In order to improve them, conditions need to be extensively screened. Besides the obvious parameters (pH, salts, precipitant, temperature, etc...), the nature of the detergents seems to be critical, and special care will be devoted to test this parameters. Crystallographic analysis of the FhuA/pb5 complex, should well-diffracting crystals be obtained, will be carried out in collaboration with W. Welte (Konstanz University, Germany).


- Single particle analysis and 2D crystallisation of FhuA-pb5 complex and different pb2 constructions (M. Chami/A. Engel, 12 months)

Purified and reconstituted FhuA grows into highly ordered crystals6. The first trials carried out in Basel show that the FhuA/pb5 complex can be reconstituted into lipid bilayers and crystallised in 2 dimensions in large but very twinned sheets.  Crystallisation conditions of the complex are different from those of FhuA and need to be improved. We will extensively screen conditions of 2D-crystallisation of the complex prepared in different detergents using different detergent removal strategies. 2D crystallisation of the different pb2 constructions will also be investigated.


Another part of the project consists in studying the straight fiber by single particle analysis. Elucidating the interactions between FhuA-pb5 and pb2 will also be investigated by single particle analysis, a method of choice to observe large multiproteic architectures. 3D reconstruction of the complex alone, and in interaction with pb2 (and pb3 and pb4 if necessary) should provide architectural insights of the FhuA-pb5-pb2 complex, and help to understand bacteriophage-host interactions.

References:

  1. A D Ferguson, E Hofmann, J W Coulton, K Diederichs, and W Welte,
    Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide, Science, 282, 2215-20 (1998).
  2. K P Locher, B Rees, R Koebnik, A Mitschler, L Moulinier, J P Rosenbusch, and D Moras,
    Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes, Cell, 95, 771-8 (1998).
  3. L. Plançon, C. Janmot, M. le Maire, M. Desmadril, M. Bonhivers, L. Letellier, and P. Boulanger,
    Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5, J. Mol. Biol., 318, 557-569 (2002).
  4. P. Boulanger, P. Jacquot, L. Plançon, M. Chami, A. Engel, C. Parquet, C. Herbeuval, and L. Letellier,
    Phage T5 straight tail fiber is a multifunctional protein acting as a tape measure and carrying fusogenic and muralytic activities, J. Biol. Chem. (2008).
  5. P. Jacquot, P. Boulanger, A. Huet, C. Ebel, M. Chami, J. Solon, P. Bassereau, and L. Letellier,
    Fusogenic and membrane activity of the C-Terminal domain of Phage T5 Straight fiber protein Pb2. Rôle in infectivity. Submitted (2008).
  6. O. Lambert, GS. Moeck, D. Levy, L. Plançon, L. Letellier and JL. Rigaud
    An 8-Å projected structure of FhuA, a ligand-gated channel of the Escherichia coli outer membrane. J. Struct. Biol 126, 145-155 (1999).