Molecular Microscopy of Membranes

MDR transporters and other membrane proteins

ABC (ATP-binding cassette) transporters are membrane transporters that hydrolyse ATP for the transmembrane transport of various amphiphilic molecules including lipids, peptides, homones, sterols, antigens. Several ABCs confer a multidrug resistance phenotype (MDR) to bacteria against antibiotics and to human against drugs used in anticancer treatments.

We have characterized by electron crystallography and single particle analysis at medium resolution the 3D architecture of BmrA, a bacterial MDR homolog of the human PgP, embedded in a lipidic membrane. We have found that in apo conformation, BmrA has a V shapped conformation with dis-engaged nucleotide binding sites. The surrounding lipid bilayer is curved around BmrA with nonsymmetrical distribution of lipids on membrane leaflets. In post-hydrolytic conformation, BmrA has a cylindrical shape and membranes are planar. This demonstrated at molecular resolution that a transmembrane protein can actively remodel membrane. Our project aims to understand this interplay between protein and the surrounding membrane.

We have also described the molecular architecture at medium resolution of ABCG2 (Breast Cancer Resistance Protein), of BmrC/BmrD, a bacterial heterodimer MDR transporter, and two MDRs from plants NtPdr1, NtPdr5.

abcweb

Fig.3D reconstruction of BmrA, a bacterial homodimeric MDR transporter in open apo conformation and embedded in membrane. The lipidic leaflet is curved around the open V shaped protein. After addition of ATP, curved membranes are transformed into planar membranes.

ABCs Related publications:

Pierman B, Toussaint F, Bertin A, Lévy D, Smargiasso N, De Pauw E, Boutry M. (2017) Activity of the purified plant ABC transporter NtPDR1 is stimulated by diterpenes and sesquiterpenes involved in constitutive and induced defenses. J Biol Chem. doi: 10.1074/jbc.M117.811935.

Toussaint F, Pierman B, Bertin A, Lévy D, Boutry M. (2017) Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells. Biochem J. doi: 10.1042/BCJ20170108.

Fribourg, P. F., Chami, M., Sorzano, C. O., Gubellini, F., Marabini, R., Marco, S., Jault, J. M., and Levy, D. (2014) 3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment. J. Mol. Biol. 426, 2059-2069.

Galián, C., Manon, F., Dezi, M., Torres, C., Ebel, C., Lévy, D., and Jault, J. M. (2011) Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2D crystallization, PlosOne 6, e19677.

Seantier, B., Dezi, M., Gubellini, F., Berquand, A., Godefroy, C., Dosset, P., Levy, D., and Milhiet, P. E. (2011) Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers, J Mol Recognit 24, 461-466.

Dezi, M., Fribourg, P. F., Di Cicco, A., Arnaud, O., Marco, S., Falson, P., Di Pietro, A., and Lévy, D. (2010) The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes, Biochim Biophys Acta. Biomembrane 1798, 2094-2101.

Orelle, C., Gubellini, F., Durand, A., Marco, S., Lévy, D., Gros, P., Di Pietro, A., and Jault, J. M. (2008) Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA, Biochemistry 47, 2404-2412.

ABCs Related grants:

ANR PRC 2016. Probing the interplay between the conformational dynamics of an ABC transporter and its membrane environment. (2016-2020). Coord. P. Bassereau. Partners D. Lévy (PCC), D. Lacoste (ESPCI), P. Tieleman (Uni. Calgary, Canada).

ANR Programme Blanc. (2006-2009) Functional and structural mechanism of multidrug ATPase pumps. Di Pietro (Coord), Partners: D. Lévy, P. Falson (IBCP, UMR 5086 CNRS/Université Lyon, Lyon), J.M. Jault (IBS, CEA-CNRS, Grenoble).

People involved in the ABC project

Daniel Lévy (PI), Su-Jin Paik (PhD Student), A. Di Cicco (IE, EM Manager); J. Manzi (IR, Biochemistry)

Collaborations:

P. Bassereau (PCC), M. Dahan (PCC, Institut Curie), E. Margeat (CBS, CNRS, Montpellier), P. Tieleman (University Calgary, Canada), S. Wilkens (University of Syracuse, USA), M. Boutry (ULB, Louvain, Belgique)

Photosynthetic core complexes, related publications (team members in bold):

During the years 2000-2007, we have combined electron crystallography, reconstitution in lipid membranes and functional studies to describe the structure of several indvidual photosynthetic complexes and the formation  supercomplexes of transmembrane proteins.

Busselez, J., Cottevieille, M., Cuniasse, P., Gubellini, F., Boisset, N., and Lévy, D. (2007) Structural basis for the PufX-mediated dimerization of bacterial photosynthetic core complexes, Structure 15, 1674-1683.

Gubellini, F., Francia, F., Turina, P., Lévy, D., Venturoli, G., and Melandri, B. A. (2007) Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: size dispersion and ATP synthase distribution, Biochim Biophys Acta 1767, 1340-1352.

Gonçalves, R., Busselez, J., Lévy, D., Seguin, J., and Scheuring, S. (2005) Membrane insertion of Rhodopseudomonas acidophila light harvesting complex 2 investigated by high resolution AFM. , J. Stuct. Biol. 149, 79-86.

Milhiet, P. E., Gubellini, F., Berquand, A., Dosset, P., Rigaud, J. L., Le Grimellec, C., and Lévy, D. (2006) High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer, Biophys J 91, 3268-3275.

Scheuring, S., Busselez, J., and Lévy, D. (2005) Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy, J Biol Chem 280, 1426-1431.

Scheuring, S., Lévy, D., and Rigaud, J. L. (2005) Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy, Biochim Biophys Acta 1712, 109-127.

Scheuring, S., Francia, F., Busselez, J., Melandri, B. A., Rigaud, J. L., and Lévy, D. (2004) Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides, J Biol Chem 279, 3620-3626.

Scheuring, S., Sturgis, J. N., Prima, V., Bernadac, A., Lévy, D., and Rigaud, J. L. (2004) Watching the photosynthetic apparatus in native membranes, Proc Natl Acad Sci U S A 101, 11293-11297.

Scheuring, S., Seguin, J., Marco, S., Lévy, D., Breyton, C., Robert, B., and Rigaud, J. L. (2003) AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2), J Mol Biol 325, 569-580.

Scheuring, S., Seguin, J., Marco, S., Lévy, D., Robert, B., and Rigaud, J. L. (2003) Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM. Atomic force microscopy, Proc Natl Acad Sci U S A 100, 1690-1693.