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Fight against mycobacterial diseases

Structural biology of mycolic acid biosynthesis in Mycobacterium tuberculosis

Our goal is to structurally characterize enzymes that were demonstrated to be essential for mycobacterial physiology. These enzymes represent potential targets for the development of new drugs for tuberculosis chemotherapy.

 

Mycolic acids, very long chain α-alkykated β-hydroxylated fatty acids, are major, specific and essential components of the mycobacterial cell envelope. Their biosynthesis is a complex process: two fatty acid synthases (FAS I and FAS II) are required to produce the so-called α-alkyl and meromycolic chains, which need to be activated before condensation can take place, finally leading after reduction to mycolates. Biosynthesis also involves a family of methyltransferases that introduce chemical modifications at two positions of the meromycolic chain.

 

 

 

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Previously, we determined the crystal structure of the mycolic acid methyltransferase Hma (J Biol Chem 2006). We next investigated the inhibition of Hma and other mycolic acid methyltransferases by cofactor analogs and found that S-adenosyl-N-decyl-aminoethyl strongly inhibits mycolic acid modification through its bisubstrate nature (J Biol Chem 2009). Hma was then used as a prototype for the development of a fragment-based drug discovery approach. A major long-term project of the group concerns the condensase Pks13. Pks13 belongs to the family of the large multifunctional type I polyketide synthases (PKS). Over the years, we have set up a strategy to elucidate the spatial arrangement of Pks13 using a combination of low- and high-resolution structural approaches (i.e. small angle x-ray scattering and x-ray crystallography) on the full length enzyme and on isolated domains or fragments. Integration of all information collected so far has allowed us to describe the molecular architecture of Pks13 at low resolution. In addition, the high-resolution crystal structure of a 52 kDa fragment containing the functionally important acyltransferase domain has been determined in different states (J Biol Chem 2012). Several other fragments of Pks13 have also been produced, crystallized, and their functional roles studied. Pks13 and other PKS need to be activated by the transfer of a phosphopantetheine arm on their acyl carrier protein domains. In M. tuberculosis, this reaction is catalyzed by the 4’-phosphopantetheinyl transferase PptT (PLoS Pathog 2012) that we have been able to produce, purify and characterize (J Struct Biol 2013), and which is currently structurally investigated. Another important partner involved in mycolic acid condensation is the fatty-acyl AMP ligase FadD32, which activates and loads the meromycolic chain onto Pks13. We have contributed to the biochemical characterization of the FadD32 enzyme from M. tuberculosis (Chem Biol 2009) and have been working on several orthologs for the development of a high-throughput screening assay for FadD32 activity (J Biomol Screen 2013) and the determination of their 3D structure.

 

  • Galandrin, S., Guillet, V., Rane, R.S., Leger, M., N, R., Eynard, N., Das, K., Balganesh, T.S., Mourey, L., Daffe, M. & Marrakchi, H. (2013). Assay Development for Identifying Inhibitors of the Mycobacterial FadD32 Activity. J Biomol Screen 18:576-87
  • Rottier, K., Faille, A., Prudhomme, T., Leblanc, C., Chalut, C., Cabantous, S., Guilhot, C., Mourey, L. & Pedelacq, J.D. (2013). Detection of soluble co-factor dependent protein expression in vivo: application to the 4’-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis. J Struct Biol 183:320-8-8
  • Bergeret, F., Gavalda, S., Chalut, C., Malaga, W., Quémard, A., Pedelacq, J.-D., Daffé, M., Guilhot, C., Mourey, L. & Bon, C. Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13. J Biol Chem 287:33675-90
  • Leblanc, C., Prudhomme, T., Tabouret, G. Ray, A., Burbaud, S., Cabantous, S., Mourey, L., Guilhot, C. & Chalut, C. (2012). 4’-Phosphopantetheinyl transferase PptT, a new drug target required for Mycobacterium tuberculosis growth and persistence in vivo. PLoS Pathog 8:e1003097
  • Léger, M., Gavalda, S., Guillet, V., Van der Rest, B., Slama, N., Montrozier, H., Mourey, L., Quémard, A., Daffé, M. & Marrakchi, H. (2009). The dual function of the Mycobacterium tuberculosis FadD32 required for mycolic acid biosynthesis. Chem Biol 16:510-190-19

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  • Vaubourgeix, J., Bardou, F., Boissier, F., Julien, S., Constant, P., Ploux, O., Daffé, M., Quémard, A. & Mourey, L. (2009). SADAE, a potent bisubstrate inhibitor of mycolic acid methyltransferases of Mycobacterium tuberculosis. J Biol Chem 284:19321-30
  • Boissier, F., Bardou, F., Guillet, V., Uttenweiler-Joseph, S., Daffé, M., Quémard, A. & Mourey, L. (2006). Further insight into S-adenosylmethionine-dependent methyltransferases: structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis. J Biol Chem 281:4434-45

 

 

Deciphering the molecular programming of polyketide synthases from pathogenic mycobacteria

Mycobacterial PKS are involved in the production of complex lipids that are important for mycobacterial cell structure and pathogenicity. Our goal is to depict the spatial arrangement of a coherent set of such megasynthases and investigate their enzymatic properties in vitro and in vivo. Because of their size and modular nature, the study of PKS represents a real challenge.

Thus, we not only work on the full-length enzymes but also on domains or fragments. In particular, we contributed to develop a new technique called “domain trapping” for screening soluble fragments of PpsC (Nucleic Acids Res 2011), which allowed determining the crystal structures of several enzymatic domains of the enzyme.

 

Beyond obtaining structural information on the selected PKS, we also aimed to describe the determinants of their specificity, pushing further the characterization of some of their domains.

 

 

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  • Pedelacq, J.-D., Nguyen, H., Cabantous, S., Mark, B.L., Listwan, P., Bell, C., Friedland, N., Lockard, M., Faille, A., Mourey, L., Terwilliger, T.C. & Waldo, G.S. (2011). Experimental mapping of soluble protein domains using a hierarchical approach. Nucleic Acids Res 39:e125

 

Mycobacterial lipid antigens presentation by CD1 proteins

CD1 proteins are specialized in the presentation of lipid antigens of both self and microbial origin to T-cell receptors. We were interested in characterizing the active complex formed between the CD1b isotype and a mycobacterial diacylsulfoglycolipid. Our initial work led us to solve the first crystal structure of natively folded human CD1b expressed from eukaryotic cells (EMBO J 2006).

 

The structure, combined with isoelectrofocalisation and native mass spectrometry, revealed that CD1b is simultaneously associated with endogenous phosphatidylcholine and a long hydrophobic spacer (diradylglycerol) molecule.

The presence of these lipids, which might play a stabilizing role and could also control adventitious antigen loading during trafficking, impeded crystallization of a CD1b-diacylsulfoglycolipid analogues complex. An in vitro activity assay was then developed and used in conjunction with isoelectrofocalisation for studying structure-activity relationships of synthetic ligands (J Immunol 2009).

In addition, native mass spectrometry and x-ray crystallography revealed that a phosphatidylcholine/antigen exchange process occurs upon antigen binding whereas the hydrophobic spacer moves within the lipid-binding groove.

This is accompanied by a structural reorganization indicating that CD1b molecular plasticity plays a crucial role for antigen presentation (Proc Natl Acad Sci U S A 2011a).

To gain insight into its function, we also determined the first crystal structure of the CD1e isotype. CD1e facilitates the processing of glycolipid antigens recognized by CD1b-restricted T cells (Science 2005).

The 3D structure of CD1e revealed a groove less intricate than in other CD1 proteins, with a wider portal (Proc Natl Acad Sci U S A 2011b). The water-exposed grooves ensure the establishment of loose contacts with lipids. CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b.

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  • Garcia-Alles, L.F., Collmann, A. Versluis, C., Lindner, B., Guiard, J., Maveyraud, L., Huc, E., Im, J.S., Sansano, S, Brando, T., Julien, S., Prandi, J., Gilleron, M., Porcelli, S.A., de la Salle, H., Heck, A.J.R., Mori, L., Puzo, G., Mourey, L & De Libero, G. (2011a). Structural reorganization of the CD1b antigen-binding groove for presentation of mycobacterial sulfoglycolipids. Proc Natl Acad Sci U S A 108:17755-60
  • Garcia-Alles, L.F., Giacometti, G., Versluis, K., Maveyraud, L., de Paepe, D., Guiard, J., Tranier, S., Gilleron, M., Prandi, J., Hanau, D., Heck, A.J.R., Mori, L., De Libero, G., Puzo, G., Mourey, L. & de la Salle, H. (2011b). The crystal structure of CD1e reveals a groove suited for lipid exchange processes. Proc Natl Acad Sci U S A 108:13230-5
  • Guiard, J., Collmann, A., Garcia-Alles, L. F., Mourey, L., Brando, T., Mori, L., Gilleron, M., Prandi, J., De Libero, G. & Puzo, G. (2009). Fatty acyl structures of Mycobacterium tuberculosis sulfoglycolipid govern T cell response. J Immunol 182:7030-37
  • García-Alles, L. F., Versluis, K., Maveyraud, L., Tesouro Vallina, A., Sansano, S., Bello, N. F., Gober, H.-J., Guillet, V., de la Salle, H., Puzo, G., Mori, L., Heck, A. J. R., De Libero, G. & Mourey, L. (2006). Endogenous phosphatidylcholine and a long spacer ligand stabilize the lipid-binding groove of CD1b. EMBO J 25:3684-92
  • de la Salle, H., Mariotti, S., Angenieux, C., Gilleron, M., García-Alles, L.F., Malm D., Berg, T., Paoletti, S., Blandine, M., Mourey, L., Salamero, J., Cazenave, J.P., Hanau, D., Mori, L., Puzo, G. & De Libero, G. (2005). Participation of CD1e in processing of microbial glycolipid antigens. Science 310:1321-4