Stage Master

Supervisors: Cécile BON, email: This email address is being protected from spambots. You need JavaScript enabled to view it., tel: 05 61 17 58 40; Célia Plisson-Chastang, email: This email address is being protected from spambots. You need JavaScript enabled to view it., tel: 05 61 33 59 50; Lionel MOUREY, email: This email address is being protected from spambots. You need JavaScript enabled to view it., tel: 05 61 17 54 36

Type I polyketide synthases (PKS) are large multifunctional enzymes responsible for the biosynthesis of a structurally diverse range of natural products with a comparably broad spectrum of biological activities. The structure elucidation of PKS is of both fundamental and applied interest, with considerable potential for structure-based engineering and drug design. Most of the current knowledge about structure-function relationships of PKS has been deduced from structures of the related type I fatty acid synthase (FAS) enzymes (1) and on combining high-resolution structural data for individual domains or didomains derived from PKS (2). Although there are not yet any atomic resolution structures of intact PKS, low-resolution models have been more recently derived from single-particle electron cryo-microscopy (3) or by hybrid approach combining X-ray crystallography and small angle X-ray scattering (4).

Pks13 is a type I polyketide synthase involved in the final step of the biosynthesis pathway – the so-called condensation step – of mycolic acids, and is essential for the viability of mycobacteria (5). It has been the focus of intensive research, at IPBS and elsewhere, aiming at characterizing its mechanism of action (6) and druggability (7, 8). In parallel with these studies, we have embarked on the structural characterization of such a complex megasynthase (186 kDa, 1733 residues comprising five catalytic domains interconnected by linker regions). Our strategy to elucidate the spatial arrangement of Pks13 was to work on the full-length enzyme and on domains or fragments, for which we have shown that their production and purification in large quantities is feasible, and to use a combination of SAXS and crystallography to provide low- and high-resolution information, respectively. For instance, the crystal structure of a 52-kDa fragment containing the acyltransferase domain has been determined in different states (9).

Our goal now is to make use of single-particle electron cryo-microscopy, which is nowadays “becoming a dominant technology” (10), to determine the atomic structure of the full-length enzyme. Preliminary results led to images of such quality that structure determination can now easily be foreseen. The objective of this internship will be to contribute to this effort through single particle reconstruction from data already available and to take part, if necessary, to grid optimization and data collection and processing on new sample preparations. It will rely on our combined fields of expertise (Mourey group: Pks13 purification, biophysics and X-ray crystallography, atomic structure interpretation; Gleizes group: cryo-EM imaging and single particle analysis).

References
1. Maier, T., Leibundgut, M., and Ban, N. (2008). The crystal structure of a mammalian fatty acid synthase. Science 321:1315-1322
2. Robbins, T., Liu, Y. C., Cane, D. E., and Khosla, C. (2016). Structure and mechanism of assembly line polyketide synthases. Curr Opin Struct Biol 41:10-18
3. Dutta, S., Whicher, J. R., Hansen, D. A., Hale, W. A., Chemler, J. A., Congdon, G. R., Narayan, A. R., Hakansson, K., Sherman, D. H., Smith, J. L., and Skiniotis, G. (2014). Structure of a modular polyketide synthase. Nature 510:512-517
4. Herbst, D. A., Jakob, R. P., Zahringer, F., and Maier, T. (2016). Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases. Nature 531:533-537
5. Portevin, D., De Sousa-D'Auria, C., Houssin, C., Grimaldi, C., Chami, M., Daffe, M., and Guilhot, C. (2004). A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc Natl Acad Sci U S A 101:314-319
6. Gavalda, S., Bardou, F., Laval, F., Bon, C., Malaga, W., Chalut, C., Guilhot, C., Mourey, L., Daffe, M., and Quemard, A. (2014). The polyketide synthase Pks13 catalyzes a novel mechanism of lipid transfer in mycobacteria. Chem Biol 21:1660-1669
7. Wilson, R., Kumar, P., Parashar, V., Vilcheze, C., Veyron-Churlet, R., Freundlich, J. S., Barnes, S. W., Walker, J. R., Szymonifka, M. J., Marchiano, E., Shenai, S., Colangeli, R., Jacobs, W. R., Jr., Neiditch, M. B., Kremer, L., and Alland, D. (2013). Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat Chem Biol 9:499-506
8. Aggarwal, A., Parai, M. K., Shetty, N., et al. (2017). Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13. Cell 170:249-259 e225
9. Bergeret, F., Gavalda, S., Chalut, C., Malaga, W., Quemard, A., Pedelacq, J. D., Daffe, M., Guilhot, C., Mourey, L., and Bon, C. (2012). Biochemical and structural study of the atypical acyltransferase domain from the mycobacterial polyketide synthase Pks13. J Biol Chem 287:33675-33690
10. Callaway, E. (2015). The revolution will not be crystallized: a new method sweeps through structural biology. Nature 525:172-174