Presentation of the Microscopic Imaging of Complex Assemblies Group (MICA)

Group leader
Irina Gutsche (CNRS Researcher)

Group members
Angélique FRAUDEAU (Lab Manager and Research Engineer)
Karine HUARD (Research Engineer)
Alister BURT (PhD Student)
Matthew JESSOP (PhD Student)
Jan FÉLIX (Postdoc)
Clarissa LIESCHE (Postdoc)
Maria BACIA (Research Engineer)
Ambroise DESFOSSES (Postdoc)
Ladislav BUMBA (visiting scientist from the Institute of Microbiology of the Czech Academy of Sciences, Prague)

from left to right: Karine, Clarissa, Jan, Irina, Matt, Alister, Angélique and Ambroise.

Activities

From the methodological point of view, the main strength of our team is single particle electron microscopy and 3D image analysis of macromolecular assemblies. Whenever possible, we also analyze our complexes and their building blocks by X-ray crystallography and small-angle scattering. In addition, in the context of modern integrated structural biology, we recently chose to start designing and performing our own biophysical and biochemical experiments in order to gain more profound insights into structural basis of molecular recognitions and regulations and to place our work in the biological context. We are increasingly fascinated by single-molecule super-resolution fluorescence microscopy and aim at combining it with cryo-electron tomography to visualize individual macromolecular assemblies in action inside the cell.

From the biological point of view, we currently head in two main research directions, the first in microbiology and the second in virology.
These projects are based on existing long-standing and already fruitful partnerships, recently enriched by new collaborations.

Microbiology project: Structure-function relationships of a unique three-component macromolecular machine at the crossroads of E. coli stress responses.

This system is based on the acid stress-inducible lysine decarboxylase LdcI that acts as antivirulence factor for several pathogens including enteroinvasive E.coli and promotes resistance of uropathogenic E.coli to fluoroquinolones in urinary tract infections. The two other components - the AAA+ ATPase RavA and its operon partner, a VWA-domain protein ViaA - are involved in oxidative stress and aminoglycoside resistance, and regulate the LdcI inhibition by the stringent response alarmone ppGpp. In collaboration with the group of Prof. Walid Houry from the University of Toronto, we solved the individual structures of LdcI and RavA by electron microscopy (EM) and X-ray crystallography, and recently unraveled the structure of the complex formed between LdcI and RavA by cryoEM. The resulting assembly forms a huge macromolecular cage which encloses a central cavity of 3x10^6 Å. In conjunction with ViaA, this ribosome-sized LdcI-RavA complex is proposed to participate in assembly and/or maturation of major respiratory complexes and to interact with other partners. We are currently performing structural and functional characterisation of this machine and its interaction with its potential targets in vitro. In parallel, we are investigating its cellular organization and regulation. This project relies on a novel partnership with the labs of Dr. Sandrine Ollagnier (BIG, Grenoble) and Prof. Frederic Barras (LCB, Marseille), and a tight cooperation with the Schoehn team and the PIXEL team (IBS). Furthermore, together with Dr. Sylvie Elsen (BIG, Grenoble), we extended the project to Pseudomonas aeruginosa. The phylogeny of the system is analyzed in collaboration with Prof. Celine Brochier-Armanet (University Lyon I).

References
A novel subfamily of bacterial AAT-fold basic amino acid decarboxylases and functional characterization of its first representative: Pseudomonas aeruginosa LdcA.
Carriel D, Garcia PS, Castelli F, Lamourette P, Fenaille F, Brochier-Armanet C, Elsen S, Gutsche I.
Genome Biol Evol. 2018 Oct 13. doi: 10.1093/gbe/evy228.

Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA.
Kandiah E, Carriel D, Perard J, Malet H, Bacia M, Liu K, Chan SW, Houry WA, Ollagnier de Choudens S, Elsen S, Gutsche I.
Sci Rep. 2016 Apr 15;6:24601. doi: 10.1038/srep24601.

Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.
Malet H, Liu K, El Bakkouri M, Chan SW, Effantin G, Bacia M, Houry WA, Gutsche I.
Elife. 2014 Aug 5;3:e03653. doi: 10.7554/eLife.03653.

The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition.
Kanjee U, Gutsche I, Ramachandran S, Houry WA.
Biochemistry. 2011 Nov 1;50(43):9388-98. doi: 10.1021/bi201161k.

Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.
Kanjee U, Gutsche I, Alexopoulos E, Zhao B, El Bakkouri M, Thibault G, Liu K, Ramachandran S, Snider J, Pai EF, Houry WA.
EMBO J. 2011 Mar 2;30(5):931-44. doi: 10.1038/emboj.2011.5.

Structure of RavA MoxR AAA+ protein reveals the design principles of a molecular cage modulating the inducible lysine decarboxylase activity.
El Bakkouri M, Gutsche I, Kanjee U, Zhao B, Yu M, Goret G, Schoehn G, Burmeister WP, Houry WA.
Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22499-504. doi: 10.1073/pnas.1009092107.

Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase.
Snider J, Gutsche I, Lin M, Baby S, Cox B, Butland G, Greenblatt J, Emili A, Houry WA.
J Biol Chem. 2006 Jan 20;281(3):1532-46.

Funding

• ANR JCJC - SVSE 8 (acidstress) 2012-2016
• GRAL Pilot Project 2014-2016
• ERC 647784 2015-2020

Virology project: Structural plasticity of negative strand RNA viruses.

Negative strand RNA viruses are at the heart of numerous human pathologies such as measles, rabies or influenza. Their genome is tightly and regularly encapsidated by the viral nucleoprotein forming nucleocapsids that provide intrinsically flexible helical templates for viral transcription and replication, and constitute attractive potential targets for antiviral drugs. In the frame of this project, our aim is to contribute to a better understanding of the structural dynamics and of the mechanism of transcription, replication and packaging of two different families of non segmented negative strand RNA viruses, i.e. paramyxoviruses and rhabdoviruses. The on-going paramyxoviral project is performed in a multidisciplinary consortium composed of Schoehn, Ruigrok and Blackledge teams (IBS) and the Blanchoin team (BIG).

References
Structural virology. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid.
Gutsche I, Desfosses A, Effantin G, Ling WL, Haupt M, Ruigrok RW, Sachse C, Schoehn G.
Science. 2015 May 8;348(6235):704-7. doi: 10.1126/science.aaa5137.

SPRING - an image processing package for single-particle based helical reconstruction from electron cryomicrographs.
Desfosses A, Ciuffa R, Gutsche I, Sachse C.
J Struct Biol. 2014 Jan;185(1):15-26. doi: 10.1016/j.jsb.2013.11.003.

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape.
Desfosses A, Ribeiro EA Jr, Schoehn G, Blondel D, Guilligay D, Jamin M, Ruigrok RW, Gutsche I.
Nat Commun. 2013;4:1429. doi: 10.1038/ncomms2435.

Nucleoprotein-RNA orientation in the measles virus nucleocapsid by three-dimensional electron microscopy.
Desfosses A, Goret G, Farias Estrozi L, Ruigrok RW, Gutsche I.
J Virol. 2011 Feb;85(3):1391-5. doi: 10.1128/JVI.01459-10.

Funding

• GRAL 2016-2018
• Fondation pour la Recherche Médicale (FRM)

Publications

The complete list of publications from Irina Gutsche can be found here.