Institut de Biologie StructuraleGrenoble / France

2019

Targeting host proteins to fight influenza

New therapeutic strategies targeting influenza are actively sought due to limitations in current drugs available. Host-directed therapy is an emerging concept to target host functions involved in pathogen life cycles and/or pathogenesis, rather than pathogen components themselves. From this perspective, researchers from the IBS/VRM group, in collaboration with the Institut Pasteur, University Paris Diderot and University Paris Descartes, focused on an essential host partner of influenza viruses, the RED-SMU1 splicing complex. They identified two synthetic molecules targeting an α-helix/groove interface essential for RED-SMU1 complex assembly. They solved the structure of the SMU1 N-terminal domain in complex with RED or bound to one of the molecules identified to disrupt this complex. They show that these compounds inhibiting RED-SMU1 interaction also decrease endogenous RED-SMU1 levels and inhibit viral mRNA splicing and viral multiplication, while preserving cell viability. Overall, their data demonstrate the potential of RED-SMU1 destabilizing molecules as an antiviral therapy that could be active against a wide range of influenza viruses and be less prone to drug resistance.

Destabilization of the human RED–SMU1 splicing complex as a basis for host-directed antiinfluenza strategy. Ashraf U, Tengo L, Le Corre L, Fournier G, Busca P, McCarthy AA, Rameix-Welti M-A, Gravier-Pelletiere C, Ruigrok RW, Jacob Y, Vidalain P-O, Pietrancosta N, Crépin T, Naffakh N. Proc Natl Acad Sci USA ;116(22):10968-10977.

How viruses release from cells after infecting them

Many cellular processes such as endosomal vesicle budding, virus budding, and cytokinesis require extensive membrane remodeling by the endosomal sorting complex required for transport III (ESCRT-III). ESCRT-III protein family members form spirals with variable diameters in vitro and in vivo inside tubular membrane structures, which need to be constricted to proceed to membrane fission. Here, researchers of the group ’Entry and Budding of Enveloped Viruses’, in collaboration with the University of Groeningen, show, using high-speed atomic force microscopy and electron microscopy, that the AAA-type adenosine triphosphatase VPS4 constricts and cleaves ESCRT-III CHMP2A-CHMP3 helical filaments in vitro. Constriction starts asymmetrically and progressively decreases the diameter of CHMP2A-CHMP3 tubular structure, thereby coiling up the CHMP2A-CHMP3 filaments into dome-like end caps. Their results demonstrate that VPS4 actively constricts ESCRT-III filaments and cleaves them before their complete disassembly. They propose that the formation of ESCRT-III dome-like end caps by VPS4 within a membrane neck structure constricts the membrane to set the stage for membrane fission.

VPS4 triggers constriction and cleavage of ESCRT-III helical filaments. Maity S, Caillat C, Miguet N, Sulbaran G, Effantin G, Schoehn G, Roos WH, Weissenhorn W. Science Advances ;5(4):eaau7198

New issue for the IBS Newsletter

Find the June 2019 issue (in french only).

ERC Advanced Grant 2019 for Martin Blackledge

The European Research Council (ERC) has awarded an "Advanced Grant" to Martin Blackledge, group leader of the Institut de Biologie Structurale (IBS - CEA/CNRS/UGA mixed research unit), for his project on the atomic resolution description of highly dynamic molecular assemblies and their role in viral replication.

Martin Blackledge is FDP group leader and Deputy Director at the IBS in Grenoble. His project entitled "DynamicAssemblies" will receive € 2.5 million financial support from the ERC over 5 years. Scientific excellence at European level is one of the main criteria for the selection of these awards dedicated to ground-breaking, high-risk projects presented by active leading Principal Investigators with a track-record of significant research achievements in the last 10 years.

Martin Blackledge studied physics at the University of Manchester and received his doctorate (D. Phil) in 1987 under the direction of Professor George Radda at the University of Oxford developing techniques for biomolecular NMR spectroscopy in vivo. In 1989 he received a Royal Society Fellowship to work at the ETH Zürich under the supervision of Professor Richard Ernst (Nobel prize for chemistry 1991) where he first started to develop methods to study biomolecular dynamics by NMR. Having discovered the beauty of the Alps, he decided to continue this work at the Institut de Biologie Structurale (CEA/CNRS/UGA) in Grenoble where he has headed the “Protein Dynamics and Flexibility by NMR” group since 2007.

The primary research interest of the Blackledge group is the study of protein dynamics by NMR, often combined with complementary biophysical techniques and advanced molecular simulation, to characterize the role of conformational flexibility in biological function on a broad range of time and length scales, from molecular recognition dynamics in folded proteins, to reorganizational dynamics of large multi-domain assemblies exhibiting extensive protein disorder to the study of fundamental physics underlying protein dynamics. He has published over 200 articles in this field. Most recently his group uses these techniques to describe highly flexible or intrinsically disordered proteins (IDPs), to map the thermodynamics and kinetics of their interaction trajectories at atomic resolution, and to determine the relationship between their dynamic behaviour and functional mechanism.

What is this project "DynamicAssemblies" about ?

IDPs are present throughout all known proteomes, playing important roles in functional mechanisms in all aspects of biology. Many molecular assemblies comprise highly dynamic components that are functionally essential. The elaboration of time-resolved, atomic resolution descriptions of the interaction trajectories of such highly disordered complexes, comprising both folded and disordered domains, is extremely challenging, requiring the development of adapted methodologies that can account for their intrinsic flexibility.
The project will in particular describe the structural and dynamic behaviour of highly disordered viral replication machines, including pre- and post-nucleocapsid assembly complexes, their interaction kinetics with host and viral partners, the effects of post-translational modifications, their assembly and functional mechanisms. The project will also identify the role of these IDPs in functional liquid droplets that provide a highly efficient means to spatially and temporally control essential molecular processes.
NMR spectroscopy is an exquisitely sensitive tool for studying highly dynamic molecular systems, allowing precise characterization of local and long-range conformational dynamics of IDPs and their complexes at atomic resolution. Ongoing development of NMR-based methods, combined with advances in fluorescence spectroscopy, cryoEM and SAS, underpinned by parallel developments in molecular simulation to ensure the necessary theoretical framework, will provide the essential tools to investigate the functional mechanisms of these previously inaccessible complexes.

Keywords
Protein dynamics, NMR, intrinsically disordered proteins, phase separation, paramyxovirus, measles, nucleocapsid, self-assembly, molecular dynamics simulation, fluorescence

Amount of the award
€2.5 million for five years

High resolution structure determination of measles nucleocapsides

Measles virus is a highly contagious human pathogen that is experiencing a dangerous resurgence throughout the world, including Europe. Replication of the virus requires encapsidation of the RNA viral genome by the viral nucleoprotein, assembling into molecular suprastructures called nucleocapsids. Researchers at the IBS have developed experimental methods (1) to encapsidate specific RNA sequences in vitro, allowing the high resolution (3.3Å) three dimensional structure determination of these nucleocapsids using cryo-electron microscopy (2). This structure reveals the positions and interactions of the RNA molecule with respect to the nucleoprotein at the highest resolution yet achieved. Using this structure, the importance of specific amino acids in the RNA binding groove for the stability of the nucleocapsids was then demonstrated using Nuclear Magnetic Resonance and site-directed mutagenesis. Crucially, this structure also determines for the first time the register of binding of the RNA genome relative to the nucleocapsid, leading to fundamental new insight into the mechanisms underpinning RNA processing by the RNA polymerase complex.

(1) Self-assembly of measles virus nucleocapsid-like particles : Kinetics and RNA sequence dependence. Milles, Jensen, Communie, Maurin, Schoehn, Ruigrok, Blackledge. Angew Chem Int Ed 55, 9356 (2016)

(2) Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication. Desfosses A, Milles S, Jensen MR, Guseva S, Colletier JP, Maurin D, Schoehn G, Gutsche I, Ruigrok RWH, Blackledge M. Proc Natl Acad Sci U S A. ; doi : 10.1073/pnas.1816417116.

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis

Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques, a collaboration of researchers show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. This study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.

The IBS-ISBG electron microscopy platform was involved in the preparation of the planctonic or cultured samples for electron microscopy imaging. An optimized sample preparation was also set up for correlative imaging between structural (TEM, SEM, FIB-SEM) and chemical imaging (X-ray fluorescence microscopy, SIMS).

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis. Decelle J, Stryhanyuk H, Gallet B, Veronesi G, Schmidt M, Balzano S, Marro S, Uwizeye C, Jouneau PH, Lupette J, Jouhet J, Maréchal E, Schwab Y, Schieber NL, Tucoulou R, Richnow H, Finazzi G, Musat N. Current Biology ; doi : 10.1016/j.cub.2019.01.073

Molecular decoding of a key step in the maturation process of heparan sulfate

Heparan sulfate belongs to the family of glycosaminoglycans, a group of negatively charged polysaccharides, present in large quantities on cell surfaces and in interstitial tissues. They exert their activities by interacting with a large number of proteins, controlling their mechanism of action and thus intervening in most of the major biological functions (morphogenesis, division, signalling and cell migration, inflammation and immune responses, angiogenesis and tissue repair,… etc.) as well as in their pathological dysfunctions. These polysaccharides comprise various glycan domains, constituting the recognition zones for heparan binding proteins and are therefore essential for "coding" the various biological functions of the molecule. The molecular mechanisms associated with the biogenesis of these domains remain poorly documented.
A collaborative project involving the laboratory Architecture et Fonction des Macromolécules Biologiques, the Institut de Biologie Structurale and the Institut de Chimie Moléculaire et des Matériaux d’Orsay, made it possible to describe the mode of action of a key enzyme in the biogenesis of heparan sulfates, the C5-epimerase, which converts glucuronic acids (GlcA) into iduronic acids (IdoA). This function is essential to the maturation process of heparan sulfate since iduronic acids are systematically present at polysaccharide interaction sites. By combining glycan engineering and chemistry, protein biochemistry and structural biology (X-ray crystallography) approaches, the residues forming the catalytic site were identified as well as the binding modes of the substrate and the product. The mechanism of action of the enzyme involves conformational changes of the polysaccharide associated with selective distortions of the glucuronic entity to be epimerized.
These results provide the molecular and mechanistic basis for new strategies to modify glucuronic/iduronic acid residues at the polymer level and to generate, by chemo-enzymatic synthesis, heparan sulfate analogues for biotechnological or therapeutic applications.

Substrate binding mode and catalytic mechanism of human heparan sulfate D-glucuronyl C5 epimerase. Debarnot C, Monneau Y R, Roig-Zamboni V, Delauzun V, Le Narvor C, Richard E, Hénault J, Goulet A, Fadel F, Vivès R R, Priem B, Bonnaffé D, Lortat-Jacob H, Bourne Y. Proc Natl Acad Sci USA published ahead of print March 14, 2019 https://doi.org/10.1073/pnas.1818333116

Cellular binding of a virus developed in cancer therapy elucidated at the atomic level

Adenoviruses cause diseases that can sometimes be fatal. By modifying them, they can also become formidable cancer cell killers. Adenoviruses are to date the most commonly used vectors in human clinical trials. Researchers have just elucidated by cryo electron microscopy the mechanism by which adenoviruses attach themselves to the cell surface. These results, published in the journal Nature Communication on March 12, 2019, could pave the way for the development of new generation anti-tumor vectors.

More than 60 adenovirus (Ad) serotypes are known in humans. While they are able to cause different types of diseases such as gastroenteritis or conjunctivitis, most of them have respiratory tropism. From childhood or adolescence, we have all been infected with several adenovirus serotypes either symptomatically (pneunomia, pharyngitis) or sometimes asymptomatically. Although not strictly speaking a major public health problem, several serotypes such as Ad3, Ad7 Ad11 and Ad14 (the subject of this study) may have been responsible for deaths among military recruits in the United States or more recently in a rehabilitation center in New Jersey where 11 of the 35 young patients died of Ad7 infection in late 2018.
In addition to this pathogenicity, adenoviruses are the most commonly used vectors in human clinical trials. Their success lies essentially in their use as oncolytic viruses. To do this, adenoviruses are modified to replicate only in the cancer cells. This treatment is already approved in China for some indications and numerous clinical trials are underway in the United States and Europe, offering great hope for new anti-tumor strategies.
Any virus needs to enter a cell to replicate, so binding to receptors on the cell surface is a key step in infection. It had been shown that some adenoviruses (Ad3, Ad7, Ad11 and Ad14) used desmoglein 2 (DSG2) to bind and enter cells. It remained to be understood at the molecular level how the adenovirus fibre (an elongated antenna-like protein present at 12 copies per virus) interacted with DSG2.
Until recently, solving the atomic structure of a small complex (the fiber/DSG2 complex is only 96kDa) seemed unthinkable. The latest technological developments of the Krios microscope have shown that this barrier can be broken. The researchers solved the structure of this complex at the atomic scale and visualized both the fibre and DSG2 residues that are involved in the interaction. Moreover, they showed that a point mutation in a single amino acid in adenoviruses was sufficient to completely abolish its binding to this receptor.
Understanding the mechanisms of adenovirus attachment to DSG2 opens two perspectives : on the one hand, consider the rational design of inhibitors of these pathogenic viruses and on the other hand, improve the targeting of oncolytic adenoviruses to tumors.

Cryo-EM structure of adenovirus type 3 fibre with desmoglein 2 shows an unsual mode of receptor engagement. Vassal-Stermann E, Effantin G, Zubieta C, Burmeister W, Iséni F, Wang H, Lieber A, Schoehn G, Fender P. Nature Communications in press, (2019)

How much one electron can do

Although living organisms are mainly composed of organic matter, many very essential natural processes depend directly on inorganic factors. The function of nearly 40% of all proteins function depends on one or more metal ions. Among the biological metal-containing cofactors, the iron-sulfur [Fe-S] clusters, ubiquitous in animals, plants and bacteria, play fundamental roles in electron transfer (respiration, photosynthesis) and the regulation of gene expression through DNA binding.
At the IBS, the Metalloproteins Unit is interested, among many others metal-containing proteins, in the bacterial metalloprotein regulator RsrR. RsrR, which coordinates a [2Fe-2S] cluter, controls the expression of certain genes involved in the redox processes of the cell. Previous studies have shown that RsrR has the particularity of modulating its binding to DNA through the reduction of its [2Fe-2S] cluster by one electron. The RsrR crystal structure shows that the cluster has a coordination previously never observed in a protein consisting of residues from two cysteines, a glutamate and a histidine. The RsrR crystals also show that the rotation of a tryptophan side chain could modulate its attachment to DNA.
This work provides the structural basis to understand how an effector as small as an electron can induce the protein-based structural changes needed for the adaptation of a bacterium to its environment.

The Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu and His Residues. Volbeda A, Pellicer Martinez MT, Crack JC, Amara P, Gigarel O, Munnoch JT, Hutchings MI, Darnault C, Le Brun NE, Fontecilla-Camps JC. J Am Chem Soc. 2019 Jan 18. doi : 10.1021/jacs.8b10823.

New insights into the recognition mechanisms of Heparan sulfate by SULF sulfatases

Through their ability to edit sulfation pattern of complex Heparan Sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, IBS resaearchers and their collaborators unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled them to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, they confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary to desulfate complex polysaccharides such as HS. In addition, they demonstrated for the first time that high affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, their findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.

Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate. Seffouh A, El Masri R, Makshakova O, Gout E, Hassoun ZEO, Andrieu JP, Lortat-Jacob H, Vivès RR. Cell. Mol. Life Sci. (2019).https://doi.org/10.{{1007/s00018-019-03027-2

A new Cryo-electron microscope for the IBS

A new cryo-electron microscope was delivered to the IBS on December 07, 2018, replacing the Polara electron microscope. This electron microscope, a ThermoFisher Glacios, has been funded by CEA, CNRS, ESRF and FRISBI.
The Glacios is an ultra-stable, state-of-the-art 200 kV, FEG electron microscope equipped with an automatic sample loading and changing system, a grid holder system compatible with Krios microscopes, a Falcon II direct electron detector coupled with an automatic data collection system (EPU) and a CETA CMOS camera. The K2 Summit direct electron detector that was installed on the Polara will also be retrofitted to this microscope in the near future. In parallel, the CETA camera will be upgraded to a "SPEED" level. Thanks to this microscope, we will be able to collect high resolution data, but also to screen grids for quality and select the best one to be transferred to an even more powerful microscope : Krios for example. This latter possibility is of special interest for the ESRF who is part of our project. It will therefore strengthen our existing collaboration with the ESRF and allow the Krios team to check some ESRF user grids on the Glacios microscope. This microscope will be used for classical "single particle" cryo imaging, cryo electron tomography and, thanks to the speed option of the CETA camera, to optimize the freezing and the data collection conditions in micro electron diffraction.
The microscope was installed in December, acceptance tests were carried out in January. The Glacios will be accessible in a service mode in March. Like for the Polara, this new microscope will be accessible directly, via FRISBI or via Instruct for national or international users.

Contacts : G. Schoehn and E. Neumann for the ISBG/IBS Cryo-electron microscopy platform, ibs-plateforme-em.contact@ibs.fr.