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A huge but elusive macromolecular cage in enterobacterial stress response as seen by cryoEM and X-ray crystallography

Abstract number: 8391

Session Code: LS06-S19

DOI: 10.1002/9783527808465.EMC2016.8391

Meeting: The 16th European Microscopy Congress 2016

Session: Life Sciences

Topic: Host-pathogen Interactions

Presentation Form: Invited Speaker

Corresponding Email: irina.gutsche@ibs.fr

Eaazhisai Kandiah (1), Hélène Malet (1), Diego Carriel (1, 2), Julien Perard (3), Maria Bacia (1), Walid A Houry (4), Sandrine Ollagnier de Choudens (3), Sylvie Elsen (2), Irina Gutsche (1)

1. IBS, MEM, Grenoble, France 2. BIG, BCI, Grenoble, France 3. BIG, LCBM, Grenoble, France 4. Department of Biochemistry, University of Toronto, Toronto, Canada

Keywords: Cryo Electron Microscopy, enteribacteria, stress response, Xray crystallography

The E. coli inducible lysine decarboxylase LdcI is an important acid stress response enzyme, whereas the AAA+ ATPase RavA is involved in multiple stress response pathways. A complex between these two proteins is thought to counteract acid stress under starvation in E. coli. We solved the crystal structure of the RavA monomer and combined it with a negative stain EM reconstruction of the RavA-ADP hexamer (El Bakkouri et al., 2010). We also determined the crystal structure of the LdcI double pentamer (Kanjee et al., 2011). While these structures provided important insights into the functions of these individual proteins, the design principles of the LdcI-RavA complex appeared even more enigmatic because it was difficult to envision how a decamer can bind a hexamer.  CryoEM analysis allowed us to fit together the pieces of the jigsaw and revealed that the LdcI-RavA complex is a surprising macromolecular cage of the size of a ribosome formed by two LdcI decamers and five RavA hexamers (Malet et al., 2014). We identified molecular determinants of this interaction and specific elements essential for complex formation, as well as conformational rearrangements associated with the pH-dependent LdcI activation and with the RavA binding (Malet et al., 2014; Kandiah et al., 2016). Moreover, we uncovered differences between the LdcI and its close paralogue, the second E. coli lysine decarboxylase LdcC thought to play mainly a biosynthetic role, finally explaining why only the acid stress response enzyme is capable of binding RavA and forming the cage. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions (Kandiah et al., 2016). Research on structure-functional relationships of this system by a combination of cryoEM, super-resolution fluorescence microscopy and other structural, biochemical, biophysical and cell biology techniques is on-going.

Kanjee, U. et al. EMBO J. 30, 931–944 (2011).

El Bakkouri, M. et al. Proc. Natl. Acad. Sci. U. S. A. 107, 22499–22504 (2010).

Malet, H. et al. eLife 3, e03653 (2014).

Kandiah, E. et al. Sci Rep. 6, 24601 (2016).

Acknowledgements

We thank Guy Schoehn for establishing and managing the cryo-electron microscopy platform and for providing training and support. We are grateful to all members of the Houry group and the Gutsche team who were or currently are involved in sample preparation and analysis for this study. For electron microscopy, this work used the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The electron microscope facility (Polara electron microscope) is supported by the Rhône-Alpes Region (CIBLE and FEDER), the FRM, the CNRS, the University of Grenoble Alpes and the GIS-IBISA. DC is the recipient of the Grenoble Alliance for Integrated Structural and Cell Biology (GRAL) PhD fellowship. This work was supported by the ANR-12-JSV8-0002 and the ERC 647784 grants to IG, the CIHR MOP-130374 to WAH, the ANR-10-LABX-49-01 and the ANR-11-LABX-0003-01.

Figures:

An overview of the LdcI-RavA cage

Conformational states of the E. coli lysine decarboxylases

Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA binding

To cite this abstract:

Eaazhisai Kandiah, Hélène Malet, Diego Carriel, Julien Perard, Maria Bacia, Walid A Houry, Sandrine Ollagnier de Choudens, Sylvie Elsen, Irina Gutsche; A huge but elusive macromolecular cage in enterobacterial stress response as seen by cryoEM and X-ray crystallography. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/a-huge-but-elusive-macromolecular-cage-in-enterobacterial-stress-response-as-seen-by-cryoem-and-x-ray-crystallography/. Accessed: December 2, 2023
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