Institut de Biologie StructuraleGrenoble / France

2019

A Unified Description of Intrinsically Disordered Protein Dynamics under Physiological Conditions using NMR Spectroscopy

Scientists estimate that one third of human proteins are intrinsically disordered proteins (IDPs), i.e. proteins without a stable three-dimensional structure. Very flexible, these biomolcules can adapt to several physiological partners and adopt a multitude of conformations. Their functioning remains poorly understood even though they play essential roles in all living organisms. Nuclear magnetic resonance (NMR) spectroscopy is ideally suited to the investigation of this behavior at atomic resolution.
In this study, measuring an extensive set of relaxation rates sampling multiple-time-scale dynamics over a broad range of crowding conditions, the researchers of the FDP group of the IBS develop and test an integrated analytical description that accurately portrays the motion of IDPs as a function of the intrinsic properties of the crowded molecular environment. In particular they observe a strong dependence of both short-range and long-range motional time scales of the protein on the friction of the solvent. This tight coupling between the dynamic behavior of the IDP and its environment allows them to develop analytical expressions for protein motions and NMR relaxation properties that can be accurately applied over a vast range of experimental conditions. This unified dynamic description provides new insight into the physical behavior of IDPs, extending their ability to quantitatively investigate their conformational dynamics under complex environmental conditions, and accurately predicting relaxation rates reporting on motions on time scales up to tens of nanoseconds, both in vitro and in cellulo.

A Unified Description of Intrinsically Disordered Protein Dynamics under Physiological Conditions using NMR Spectroscopy. Wiktor Adamski, Nicola Salvi, Damien Maurin, Justine Magnat, Sigrid Milles, Malene Ringkjøbing Jensen, Anton Abyzov, Christophe Moreau, Martin Blackledge. Journal of the American Chemical Society ; 141(44):17817-17829

Structural basis for broad HIV-1 neutralization by the MPER-specific human Broadly neutralizing antibody LN01

The key to HIV vaccine development is the induction of broadly neutralizing antibodies (bnAb). The currently known bnAbs are directed against six regions of the HIV envelope glycoprotein trimer composed of gp120 and gp41. Several bnAbs that target a highly conserved epitope named "MPER" located on gp41 have been identified. Candidates for the development of a vaccine based on peptides mimicking this epitope have been tested without success. What is the reason for these failures ? In this study, researchers from EBEV group at IBS in collaboration with several universities characterized a novel highly potent human bnAb (LN01) directed against the MPER epitope of HIV. LN01 neutralizes 92% of a multi-clade 118 primary virus panel. By examining the structural details of LN01 epitope recognition, the researchers showed that the MPER epitope extends into the transmembrane region, which is essential for LN01 gp41 recognition. The structural studies revealed that the MPER epitope forms a continuous helix with the transmembrane region (TM) and identified two lipid binding sites. Based on molecular dynamics simulation, the researchers propose a model of interaction of LN01 with its epitope inserted in the viral membrane thereby allowing LN01 interaction with lipids. Both epitope and lipid interaction have been shown to be important for virus neutralization. All these data demonstrate that a candidate for vaccine development must include the MPER and transmembrane domain of gp41 correctly inserted into a lipid bilayer to induce broadly, cross-clade neutralizing antibodies.

Structural basis for broad HIV-1 neutralization by the MPER-specific human Broadly neutralizing antibody LN01. Pinto D, Fenwick C, Caillat C, Silacci C, Guseva S, Dehez F, Chipot C, Barbieri S, Minola A, Jarrossay D, Tomaras GD, Shen X, Riva A, Tarkowski M, Schwartz O, Bruel T, Dufloo J, Seaman MS, Montefiori DC, Lanzavecchia A, Corti D, Pantaleo G and Weissenhorn W. Cell Host Microbe ; 26(5):623-637.e8.

Paoletti Prize 2019 for Sigrid Milles (IBS/FDP)

On the 30st of Octobre 2019, Sigrid Milles received the Paoletti Prize 2019 for her work on intrinsically disordered proteins studied by single molecule fluorescence and nuclear magnetic resonance spectroscopy.
This prestigious award is presented in memory of Claude Paoletti, former chief scientist of the CNRS Life Sciences Department who took many initiatives to support young researchers.

Official opening of the 2019 Science Festival in Isere on EPN Campus

Thursday 03 October saw the official opening of the 2019 Science Festival on the EPN Campus.

The launching of the 2019 Science Festival in Isere began with visits of the NMR and electron microscopy platforms of the IBS, the ESRF tunnel, the EMBL-IBS partnership platform for high throughput protein crystallography, the Science building and two ILL-ESRF joint laboratories were proposed.

Then the opening ceremony in the IBS seminar room was attended by Chloé Lombard, representing Mr Préfet de l’Isère, Viviane Henry, representing Madam Rector of the Academy of Grenoble, Christophe Ferrari, the President of Grenoble Alpes Metropole, Eric Piolle, Mayor of Grenoble, and Jeany Jean-Baptiste, Director of la Casemate. The directors of the four EPN institutions (EMBL, ESRF, IBS, ILL) and people in charge of the Science Festival in other academic laboratoires or associations were also present.

Visite de la plateforme de Microscopie electronique de l’IBS - © IBS
Visite de la plateforme de RMN de l’IBS - © IBS
Visite du tunnel ESRF - © ESRF
Cérémonie de lancement - © ILL
M. Weissenhorn, directeur de l’IBS - © IBS
Mme Jeany Jean-Baptiste, Directrice de la Casemate - © IBS
M.Eric Piolle, maire de Grenoble - © IBS
M.Christophe Ferrari, Président, Grenoble-Alpes-Métropole - © IBS
Mme Viviane Henry représentant Madame la Rectrice de l’Académie de Grenoble - © IBS
Mme Chloé Lombard, représentant Monsieur le Préfet de l’Isère - © IBS

It was a great opportunity to highlight our cooperative research facilities and our commitment to the promotion and dissemination of science.
More information about IBS events for the 2019 Science Festival can be found here.

A chimeric pseudo-adenovirus to combat emergent diseases

Infectious diseases continue to decimate populations around the world. Among the means at our disposal to counter these threats, vaccination has proven to be exceptionally powerful. Smallpox has been eradicated, measles and polio are controlled by vaccination. However, serious threats still exist, as evidenced by epidemics caused by the Ebola or Zika viruses. Another recent example is the Chikungunya virus, a viral pathogen transmitted by the bite of a tiger mosquito. Once confined to sub-Saharan Africa, the Chikungunya virus has recently spread worldwide as these mosquito vectors move geographically up to the poles as a result of climate change.
The "Adenovirus" team of the Methods & Electron Microscopy group at IBS has formed an international consortium with EMBL and the University of Bristol to develop a new vaccine technology. Indeed, this team is working on an adenovirus protein that spontaneously self-assembles by 60 to give a particularly stable particle even without refrigeration, similar to a virus but not infectious. A cryo electron microscopy study showed that this particle has a very flexible quasi-spherical surface. Engineering of this adenoviral protein was then carried out to customarily replace these exposed regions with those from other pathogens. Proof of principle was established by expressing a particle displaying neutralizing epitopes of the Chikungunya virus. These chimeric neo-particles Adenovirus/Chikungunya have shown promising results in animal studies as shown both by their drainage to the lymph nodes and the humoral response produced against the epitopes of the Chikungunya virus covering the particle. This easy-to-use vaccine technology, based on a single particle that can be modified by synthetic biology, has been patented by the CNRS and EMBL and could eventually be used to fight many other infectious diseases. The results have just been published in the prestigious journal Science Advances.

Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display. Charles Vragniau, Joshua C. Bufton, Frédéric Garzoni, Emilie Stermann, Fruzsina Rabi,Céline Terrat, Mélanie Guidetti, Véronique Josserand, Matt Williams, Christopher J. Woods, Gerardo Viedma, Phil Bates, Bernard Verrier, Laurence Chaperot, Christiane Schaffitzel, Imre Berger and Pascal Fender. Science Advances ; Vol. 5, no. 9, eaaw2853

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

Given their complexity, the structure determination of high molecular weight biological machinery often remains a challenge for experimental methods available to structuralists. Researchers from the MEM, NMR and DYNAMOP groups at IBS, in collaboration with the University of Frankfurt and the NIH, have developed an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å. The resulting structure exceeds current standards of NMR and EM methods in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available and thus opens new perspectives for solving the structure of complex biological systems.

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex. Gauto DF, Estrozi LF, Schwieters CD, Effantin G, Macek P, Sounier R, Sivertsen AC, Schmidt E, Kerfah R, Mas G, Colletier JP, Güntert P, Favier A, Schoehn G, Schanda P, Boisbouvier J. Nature Communications ;10(1):2697

Insights into the movements of aromatic residues in a 0.5 MDa enzyme by solid-state NMR

Aromatic residues play key roles in many proteins, and are involved in protein-ligand and protein-protein interactions. They also can be found in the hydrophobic core of proteins, where they are crucial for protein stability. For these reasons, studying their dynamics can provide rich information on reaction mechanisms and folding. Although solution-state NMR has been used to study the motions of aromatic residues for decades, these studies have been limited to small proteins, due to inherent physical restrictions. A new approach developed by the IBS NMR group, in collaboration with Japanese and Austrian chemists, makes it possible to gain insights into the movements of aromatic residues even in very large proteins. The approach combines solid-state NMR and specific isotopic labelling of phenylalanines or tyrosines. The study by Gauto et al applied this method to the 468 kDa enzyme TET2 protein and demonstrated, among other things, the rotational kinetics of phenylalanines over a wide temperature range, down to -170°C. Temperature-dependent measurements reveal how the motions of the protein are activated when the temperature is increased, with an interesting appearance of motion at about -70 °C, often call the "glass-transition temperature". Furthermore, the approach allowed to infer "invisible" states with life times of microseconds, which, interestingly cluster around a pore located between subunits. This methodology also allows to obtain distance information between aromatic residues and their environment, which are valuable for determining the three-dimensional structures of proteins.

Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H-13C Labeling and Fast Magic-Angle Spinning NMR. Gauto DF, Macek P, Barducci A, Fraga H, Hessel A, Terauchi T, Gajan D, Miyanoiri Y, Boisbouvier J, Lichtenecker R, Kainosho M, Schanda P. Journal of the American Chemical Society ; 141(28):11183-11195

Mechanism of allosteric activation of an enzyme by an inhibitor

The finding that an inhibitor activates an enzyme appears counterintuitive. How can an inhibitor, which binds to the active site, increase the enzyme’s activity ? In this study, researchers at IBS (NMR, MICA and PG groups), in collaboration with colleagues in Nancy and Zaragoza, revealed how the 300 kDa large protease ClpP undergoes this intriguing allosteric activation by an inhibitor as well as by substrates. A multi-technique approach, involving X-ray crystallography, solution- and solid-state NMR, molecular dynamics simulations and calorimetry, shows that substoichiometric binding of inhibitors to active sites shifts the equilibrium in this oligomeric protein to a more active extended state. The findings may provide new routes for the development of drugs of this potential antibiotic protein target.

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors. Felix J, Weinhäupl K, Chipot C, Dehez F, Hessel A, Gauto DF, Morlot C, Abian O, Gutsche I, Velazquez-Campoy A, Schanda P, Fraga H. Science Advances ; Vol. 5, no. 9, eaaw3818

Cell morphology and nucleoid dynamics in dividing D. radiodurans.

In this study, IBS researchers (VIC group/Timmins team in collaboration with DYNAMOP group/ Bourgeois team) demonstrated through conventional fluorescence microscopy and super-resolution approaches, the organization and dynamics of the nucleoid of Deinococcus radiodurans, a bacterium well known for its outstanding radiation resistance. This work revealed for the first time that bacterial nucleoids are complex entities that change structure as a function of the cell cycle and can adopt more or less compact conformations. The HU protein seems to play a key role in this process.

Cell morphology and nucleoid dynamics in dividing D. radiodurans. Floc’h K, Lacroix F, Servant P, Wong YS, Kleman JP, Bourgeois D and Timmins. Nature Communications ; 10(1):3815

3.3 Å resolution structure of Hantaan virus nucleocapsid revealed by cryo-EM

The virus Hantaan, that belongs to the Hantaviridae family (Bunyavirales order) and is transmitted by rats and mice, can give rise in human to fatal hemorrhagic fevers against which no treatment is currently available. The genomic segments of this negative strand RNA virus are surrounded by multiple copies of nucleoproteins that together form helical nucleocapsids. The Methods and Electron Microscopy Group used state-of-the art cryo-electron microscopy to determine the 3.3 Å resolution structure of recombinant non-pathogenic Hantaan virus nucleocapsid, thereby revealing how nucleoproteins interact to form nucleocapsids that contain a continuous groove able to encompass and protect the RNA genome. This work greatly beneficiated from the IBS state-of-the-art cryo-EM platform and from the high-end Titan Krios ESRF cryo-electron microscope.

High resolution cryo-EM structure of the helical RNA-bound Hantaan virus nucleocapsid reveals its assembly mechanisms. Arragain B, Reguera J, Desfosses A, Gutsche I, Schoehn G, Malet H. Elife ; 8. pii : e43075.

Importance of lncRNA tertiary structure in key cellular processes

There are several cellular mechanisms to fight cancer. One of the major actors is the p53 protein which, when it becomes inactive, greatly increases the risk of cancer development. Maternally expressed 3 (MEG3), a long non-coding RNA (lncRNA, >500 kDa), is another molecule that prevents cancer through its stimulating activity on p53.
The results of a study on the structure of MEG3, published in Molecular Cell, could help to advance the diagnosis and treatment of certain types of cancer. It was led by Marco Marcia’s group (EMBL, Grenoble) in collaboration with the AFM team of the Institute of Structural Biology (IBS, Grenoble), the CIBIO department (University of Trento, Italy) and the Max Delbrück Center (Berlin).
This study shows that it is through a precise three-dimensional structure that MEG3 ensures its cellular function, in particular through very specific RNA structures known as "kissing loops".
The IBS AFM team determined by single lncRNA molecule imaging, the conformation of MEG3 under several experimental conditions (native, destabilizing, and denaturing). The attached figure provides a sample of 100 isolated molecules of native MEG3 deposited on mica and air-imaged by atomic force microscopy (AFM) with Peak-Force mode. Compared to AFM imaging of unstructured (poly-A) or known to be structured (group II intron) RNA molecules, the results demonstrate the presence of a particular folding for MEG3 which is consistent with the biochemical and biophysical results of this study.

Conserved pseudoknots in lncRNA MEG3 are essential for stimulation of the p53 pathway. Uroda T, Anastasakou E, Rossi A, Teulon J-M, Pellequer J-L, Annibale P, Pessey O, Inga A, Chillon I and Marcia M. Mol. Cell 75 : 1-14.

A strategy to reduce fluorescence intermittencies in sptPALM

Super-resolution microscopy makes it possible to observe living matter at the nanoscopic scale, not only from a structural point of view but also from a dynamic point of view. In the latter case, individual target molecules are monitored as they diffuse into a cell : this is the "sptPALM" technique (single-particle-tracking localization microscopy). However, a major obstacle to this technique is the imperfection of the fluorescent markers used to label the target molecules. These markers are most often photoconvertible fluorescent proteins (PCFPs). In particular, PCFPs have a tendency to "blink", i.e. to temporarily shut down, which quickly causes the loss of the individual molecules tracks.
In this work, carried out in collaboration with researchers from the Catholic University of Leuven in Belgium, researchers from IBS/DYNAMOP studied the origin of the blinking phenomenon in the case of the photoconvertible fluorescent protein "mEos4b". A "dark" (non-fluorescent) state was highlighted, and its detailed characterization revealed a high sensitivity to cyan-coloured light. Thus, a low illumination of the sample with a laser at 488 nm effectively depopulates this dark state, forcing a rapid return to the fluorescent state and considerably reducing the intensity of the blinking. In sptPALM, this additional illumination at 488 nm is very easy to achieve, providing a significant improvement in data quality with minimal effort.

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM. De Zitter E, Thédié D, Mönkemöller V, Hugelier S, Beaudouin J, Adam V, Byrdin M, Van Meervelt L, Dedecker P and Bourgeois D. Nature Methods ; volume 16, pages707–710.

Mechanism for assembling pores on the bacterial surface : a strategy for secreting toxins

Bacteria have developed different secretion systems that allow them to secrete toxins to the outside of the cell. These machines are important for infection, colonization and microbial communication processes. An important element of many of these machines is secretin, a membrane protein that forms a pore on the bacterial surface allowing toxins to escape. Using cryo electron microscopy, crystallography, and microbial genetics techniques, the MEM and PATBAC groups of IBS, in collaboration with the University of Saskatchewan in Canada, solved the structure (at approximately 3.5 Å resolution) of two secretins from the emerging pathogens Vibrio vulnificus and Aeromonas hydrophila and characterized the mechanism for their assembly on the bacterial membrane. This work has shown that the assembly of some secretins requires the help of membrane lipo-proteins called "pilotins", while others are assembled independently. Since the secretin-pilotin interface is essential for the virulence of many pathogens, it could be an original target for the development of new inhibitors of infections caused by bacteria.

Structure and assembly of pilotin-dependent and -independent secretins of the Type II secretion system. Howard SP, Estrozi L, Contreras-Martel C, Bertrand Q, Job V, Martins A, Schoehn G, Dessen A. PLoS Pathogens ; 15(5):e1007731

Double labelling to facilitate the study of glycosaminoglycans

Heparan sulfate (HS) are sulfated polysaccharides of the Glycosaminoglycans family that participate in many cellular processes because of their ability to interact and modulate a wide range of signalling proteins. These interactions involve particular HS motifs, defined by their saccharide sequence and sulfation profile. However, the structural features of these functional domains remain mostly unknown, due to the molecular complexity of these polysaccharides and a lack of tools dedicated to their analysis.

In this context, the researchers of the SAGAGAG group, in collaboration with the Parisian Institute of Molecular Chemistry and the LG2A laboratory in Amiens, have developed a method allowing a double labelling of HS oligosaccharides, using Thia-Michael type addition and deuterium incorporation, respectively at the non-reducing and reducing ends of the sugar. This new labelling technique allows the combination of microgram-scale oligosaccharide labelling and mass spectrometric analysis, without altering HS/protein recognition properties, as demonstrated for heparinase I and HSulf-2 enzymes. This method is a new tool that should allow new developments for the sequencing of GAG oligosaccharides and the elucidation of new structure/function relationships.

A microscale double labelling of GAG oligosaccharides compatible with enzymatic treatment and mass spectrometry. Przybylski C, Bonnet V, Vivès RR. Chemical Communications ; 55(29):4182-4185.

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

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.