Institut de Biologie StructuraleGrenoble / France

2020

Towards the mechanism of action of the mitochondrial Complex I assembly complex

The respiratory complexes located in the inner membranes of our mitochondria are true macromolecular batteries : they couple the flow of electrons through a wire of metal clusters and co-factors with proton transfer to create a gradient that provides the energy needed to produce ATP and thus power the essential processes of life. The first complex in the respiratory chain, called Complex I, is one of the largest membrane proteins, composed of 45 subunits. The processes involved in assembly of Complex I and its sophisticated regulation are still poorly understood, but it is known that their disruption leads to neurogenerative diseases such as Alzheimer’s and accompanies the ageing process in general. In this manuscript, resulting from a collaboration between the Montserrat Soler-Lopez team at ESRF and researchers at IBS, a combination of biochemical, biophysical and structural techniques was used to elucidate the mechanism of action of a sub-complex of the human Complex I assembly complex. The ECSIT protein was found to play a key role in the regulation of the ACAD9 protein which switches between two incompatible activities. Indeed, the binding of ECSIT to ACAD9 ejects the FAD cofactor necessary for the involvement of ACAD9 in fatty acid oxidation, in order to redirect it towards action in Complex I assembly. These results are of great relevance to the field of mitochondrial neurobiology, and open multiple research avenues.

Assembly of the mitochondrial Complex I assembly complex suggests a regulatory role for deflavination. Giachin G, Jessop M, Bouverot R, Acajjaoui S, Saidi M, Chretien A, Bacia-Verloop M, Signor L, Mas PJ, Favier A, Borel Meneroud E, Hons M, Hart DJ, Kandiah E, Boeri Erba E, Buisson A, Leonard G, Gutsche I and Soler-Lopez M. Angewandte chemie 2020 ; doi : 10.1002/anie.202011548

Contact : Irina Gutsche (Microscopic Imaging of Complex Assemblies Group)

Structural and functional investigations of novel microbial rhodopsins

Microbial rhodopsins constitute a large and diverse superfamily of light-sensitive membrane proteins. They play a major role in solar energy capture in the sea, utilizing it to perform ion translocation, sensory, enzymatic, and other activities. Microbial rhodopsins also found essential applications in medicine in neuroscience, being the core of optogenetics - biotechnology for optical control on living cells and tissues. Our group studies the functional and structural properties of new families of rhodopsins. Recently, we reported the first high-resolution insights into the heliorhodopsin family (1), which showed that these proteins are likely unique photoenzymes with unusual architecture. Next, we determined the structural basis of light-driven sodium pumping by solving the structure of KR2 rhodopsin in its active state (2). Finally, we characterized two members of viral rhodopsins group 1 (3). We showed that they are light-gated Na+/K+-selective channels, inhibited by Ca2+. We also solved a structure of one of them at 1.4 Å. Thus, our studies revealed both functional and mechanistic features of the distinct clades of microbial rhodopsins but also created a strong fundament for further investigations of the expanding superfamily.

(1) High Resolution Structural Insights into the Heliorhodopsin Family. Kovalev K., Volkov D, Astashkin R, Alekseev A, Gushchin I, Haro-Moreno JM, Rogachev A, Balandin T, Borshchevskiy V, Popov A, Bourenkov G, Bamberg E, Rodriguez-Valera F, Bueldt G, Gordeliy V. Nature Communications 2020 Feb 25 ;11:2137.

(2) Molecular mechanism of light-driven sodium pumping. Kovalev K, Astashkin R, Gushchin I, Orekhov P, Volkov D, Zinovev E, Marin E, Rulev M, Alekseev A, Royant A, Carpentier P, Vaganova S, Zabelskii D, Baeken C, Sergeev I, Balandin T, Bourenkov G, Carpena X, Boer R, Maliar N, Borshchevskiy V, Bueldt G, Bamberg E, Gordeliy V. Nature Communications 2020 May 1 ; 11:2137.

(3) Viral Rhodopsins 1 : A Unique Family of Light-Gated Ion Channels. Zabelskii D, Alekseev A, Kovalev K, Rankovic V, Balandin T, Soloviov D, Bratanov D, Savelyeva E, Podolyak E, Volkov D, Vaganova S, Astashkin R, Chizhov I, Yutin N, Rulev M, Popov A, Eria-Oliveira AS, Rokitskaya T, Mager T, Antonenko Y, Rosselli R, Armeev G, Shaitan K, Vivaudou M, Büldt G, Rodriguez-Valera F, Kirpichnikov M, Moser T, Koonin E, Offenhäusser A, Bamberg E, Gordeliy V. Nature Communications 2020 Nov 11 2020 ;11(1):5707.

Contact : Valentin Gordeliy

How do chaperones that they transport other proteins into mitochondria distinguish between their different "cargo proteins" ?

The human mitochondrial proteome is estimated to contain more than a thousand proteins, 99% of which are synthesized outside the mitochondria. These proteins are imported inside the mitochondria in very specific places, in one of the membranes, in the inter-membrane space or in the matrix. The molecular mechanisms of this protein import are still poorly understood at the atomic level. A study by the NMR group has shed light on the mechanistic basis of the specificity of the chaperone system of the intermembrane space of the mitochondria. Sucec et al. studied how two chaperones which are structurally homologous but have different functions, interact with different membrane-protein precursors. By combining different techniques including NMR, Small Angle X-ray Scattering (SAXS), Analytical Ultracentrifugation (AUC) and Molecular Dynamics simulations and other biophysical / biochemical approaches, they were able to demonstrate the formation different types of complex chaperones. The delicate balance between promiscuity and specificity that these chaperones must satisfy is the result of a combination of hydrophobic and hydrophilic interactions towards different client proteins. This work contributes to the advancement of knowledge on the biogenesis of mitochondria, and more generally on the functioning of chaperone proteins, important players in the homeostasis of cellular proteins.

Structural basis of client specificity in mitochondrial membrane-protein chaperones. Sucec I, Wang Y, Dakhlaoui O, Weinhaupl K, Jores T, Costa D, Hessel A, Brennich M, Rapaport D, Lindorff-Larsen K, Bersch B and Schanda P. Science Advances 2020 ;6(51):eabd0263

Contact : Paul Schanda (Biomolecular NMR Spectroscopy group)

The HU protein of Deinococcus radiodurans imaged by AFM

Unlike eukaryotes, which have their genetic material within a nucleus, bacterial DNA is now known to be rather packed in diffuse, but not random, regions called nucleotides. But how are they organized ?
HU is an important, even essential, protein in some bacterial nucleotides. However, the cellular function of HU in DNA compaction has been controversial practically since its identification in the 1970s, notably because of its different behavior from one species to another. The AFM team of the MEM group, in collaboration with the DNA Lesions and Repair team of the VIC group, has made it possible to image for the first time the HU protein of Deinococcus radiodurans (DrHU) by Atomic Force Microscopy (AFM). This work highlights a brand new AFM image processing, based on a filter using the Laplacian operator, which allows the DrHU protein to be observed on native plasmids produced by E. coli as well as on generated linearized plasmids. This filter improves the visibility of single molecules beyond the limitation related to the AFM tip-sample convolution phenomenon. AFM images suggest two major functions of DrHU in the condensation, but also the decondensation of double-stranded DNA. The self-compacting dynamics of naked DNA, as well as the concentration of DrHU, are important parameters in the cellular performance of DrHU.

Nanoscale surface structures of DNA bound to Deinococcus radiodurans HU unveiled by atomic force microscopy. Chen SWW, Banneville AS, Teulon JM, Timmins J and Pellequer JL. Nanoscale 2020 ;12(44):22628-22638

Contact : Jean-Luc Pellequer (Methods and Electron Microscopy group)

Paul Schanda receives Varian Young Investigator Award

The EUROMAR committee awarded the Varian Young Investigator prize to Paul Schanda (IBS/NMR). This prestigious award was created in honour of Russel Varian, a pioneer in NMR spectroscopy, to recognize a single investigator within the first 12 years of his/her independent career, for achievements in any area of magnetic resonance.
This award recognises Paul Schanda’s methodological developments to characterise the dynamics and structure of proteins in solid and liquid states. Some of the methods developed by his team are widely used to monitor, for example, the kinetic processes of proteins, or to detect transient conformational states (details).
A few weeks ago, Paul Schanda was also awarded the ICMRBS Founders’ Medal, which recognises the outstanding contributions of young scientists to the development of magnetic resonance in biological systems.

IBS in the new health context

Our activities are maintained subject to organizational adjustments : people involved in laboratory-based research activity work on site but teleworking is maximised whenever possible.
Staff on site are reminded to respect strictly sanitary measures and reduce social interactions.
All scientific events such as meetings and seminars are hold online.

SARS-CoV-2 : discovery of a new mode of transmission

Glycoprotein S, located on the SARS-CoV-2 virus membrane, is known to allow the entry of this virus into human cells via the ACE2 receptor, present on the surface of infected cells. The Membrane & Pathogens group, in collaboration with IBS/IRPAS and MEM groups and groups from Spain and Italy, have discovered that C-type lectin receptors (CLRS) of antigen-presenting cells are involved in the overall infection process. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. After attaching the virus to the cell, these lectin receptors are capable of promoting virus transfer to permissive ACE2+ cells. It is therefore a new mode of transmission in the infection process, detailed in an article posted on the BioRxiv preprint site (doi.org/10.1101/2020.08.09.242917) which is currently under evaluation by a peer-reviewed journal. They have also shown that it is possible to inhibit this new mode of virus transmission through the use of glycomimetics (sugar mimetics created by organic synthesis) previously developed at IBS.

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist. Thépaut M, Luczkowiak J, Vivès C, Labiod N, Bally I, Lasala F, Grimoire Y, Fenel D, Sattin S, Thielens N, Schoehn G, Bernardi A, Delgado R, Fieschi F. doi : https://doi.org/10.1101/2020.08.09.242917

Contact : Franck Fieschi, UGA professor attached to the IBS (Membrane & Pathogens group)

Cryo-EM structure of a key enzyme in action gives insights into the replication of a human pathogenic virus

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening human pathogens for which there is currently no treatment (La Crosse virus, Hantaan virus, Crimean Congo virus, Lassa virus). The replication and transcription of their genome are essential steps of their viral cycle and are catalyzed by a key viral enzyme : the RNA-dependent RNA polymerase. The MEM group at IBS, in collaboration with Dr. Cusack’s group at EMBL Grenoble, describes here the structure of the complete RNA-polymerase of the La Crosse virus obtained at 3 Å resolution by cryo electron microscopy, using data collected on the Glacios cryo-microscopes from IBS and Krios from ESRF. This structure reveals the position and organization of the C-terminal part of the RNA polymerase which includes a cap-binding domain necessary for transcription initiation. Two states could be visualized, pre-initiation and elongation. In particular, this allows to highlight the conformational changes necessary for the formation of a double-stranded 10-base pair RNA in the active site cavity during elongation. The structural details and dynamics of the functional elements identified provide mechanistic insight into bunyavirus transcription and may be crucial for the future development of RNA polymerase inhibitors.

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational change. Benoît Arragain, Grégory Effantin, Piotr Gerlach , Juan Reguera , Guy Schoehn, Stephen Cusack, Hélène Malet. Nature Communications 2020 ;11(1):3590. doi : 10.1038/s41467-020-17349-4.

Contact : Hélène Malet

Following protein aggregation in real time by neutron spectroscopy

Protein aggregation into amyloid superstructures is the molecular manifestation of a large variety of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Evidence is increasing that transient on-pathway oligomers are actually the toxic species, such that time-resolved monitoring of protein aggregation is highly desirable. Whereas time-resolved structural techniques have been developed and applied to study protein aggregation, methods accessing protein center-of-mass and internal diffusion dynamics remain only sparsely available. Yet, changes in protein dynamics have been postulated to play an essential role in driving and accompanying protein aggregation.
Scientists of the Institut Laue Langevin, the Institut de Biologie Structurale and the University of Copenhagen developed a time-resolved version of incoherent neutron scattering experiments on IN16B to follow protein aggregation and applied it to study the assembly of lysozyme in aqueous solution into particulate superstructures. Surprisingly, the internal protein dynamics on the nano-to picosecond time scale does not change during the entire aggregation process. The center-of-mass diffusion, on the other hand, decreases as aggregation proceeds and can be well explained by a single exponential process. By complementing the neutron results with fluorescence measurements, electron microscopy, infrared spectroscopy, x-ray powder diffraction, and dynamic light scattering, a comprehensive picture was painted in which lysozyme particulate formation is a one-step process with protein backbone and side chain dynamics remaining unchanged throughout aggregation.
The work establishes a framework to follow protein aggregation quantitatively in real time at a molecular level, simultaneously accessing center-of-mass and internal diffusivities, which will be invaluable for addressing pathological pathways of protein aggregation. This framework is not limited to proteins, but can be applied to macromolecules in general to study a large variety of processes such as self-assembly and emerging aggregates and crystals.

Tracking Internal and Global Diffusive Dynamics During Protein Aggregation by High-Resolution Neutron Spectroscopy. Pounot K, Chaaban H, Foderà V, Schirò G, Weik M, Seydel T. J.Phys.Chem.Lett. 11, 15 (2020)

Contact : Martin Weik

Hélène Malet appointed Junior Member of the Institut Universitaire de France

Hélène Malet, Associate Professor at the University Grenoble Alpes and research scientist in Dr. Guy Schoehn’s Methods and Electron Microscopy group at IBS, is appointed Junior Member of the Institut Universitaire de France (IUF) from October 1, 2020 for a period of five years.

Viral replication and transcription are key steps of the viral cycle. Hélène Malet analyses the structure of viral proteins involved in these processes, in particular viral polymerases. During her thesis, carried out under the supervision of Dr. Bruno Canard at the AFMB, Marseille, she characterized by X-ray crystallography a polymerase structure of the Flaviviridae family, to which the Dengue virus belongs. Then, eager to learn a complementary method of structural biology, she did a post-doctorate in electron microscopy in the laboratory of Pr. Helen Saibil at Birkbeck College, London. Since then, she combines her interest in electron microscopy and viral replication. She undertook a post-doctoral fellowship on the structural analysis of Peribunyaviridae polymerase in the group of Dr. Stephen Cusack at EMBL Grenoble, before being recruited as an UGA Associate Professor at IBS in the team of Dr. Guy Schoehn in the Electron Microscopy and Methods group.

Her research project focuses on the structural and functional analysis of bunyavirus replication, a viral order consisting of many highly pathogenic human viruses against which no drugs or vaccines are available. The latest advances in electron microscopy and the presence of state-of-the-art electron microscopes at IBS and ESRF enable to determine high-resolution structures of these essential enzymes thereby revealing their modes of action, a key step for the future development of anti-virals. In the longer term, this project aims to understand the mechanisms of interactions between viral proteins and host proteins involved in the regulation of viral replication, combining high-resolution electron microscopy of isolated particles and cellular electron microscopy, allowing an integrative view of these processes. This project is financially supported by the ANR (HiPathBunya) and will make an extensive use of IBS technology platforms managed by ISBG and funded by FRISBI and Gral. The appointment at the IUF will allow her to devote more time to this project.

Molecular insight into the transmission of avian influenza to enable human infection

IBS ​Researchers, in collaboration with EMBL Grenoble, have used NMR to reveal the molecular mechanisms that support the adaptation of avian influenza virus from birds to humans.
In birds, the avian influenza virus acts via the interaction of its polymerase with a key transcription factor : ANP32A. Two regions of these highly dynamic molecules seemed to be strongly implicated, but the molecular basis of the interaction remained unknown, preventing researchers from understanding how certain mutations in the viral polymerase enabled it to adapt from bird to human and thus acquire a capacity for cross-species contagion. These highly dynamic, so-called intrinsically disordered molecular regions do not form unique structures, nor do they crystallize, complicating their study.
Using NMR, researchers at the Institut de Biologie Structurale have succeeded in deciphering these mechanisms : in birds, the so-called "627-NLS domain" of the viral polymerase binds to a specific area, involving a unique sequence, of the transcription factor ANP32A. However, this sequence does not exist in the human version of the transcription factor - the viral polymerase therefore needs to develop an alternative strategy for binding the transcription factor if it is to successfully infect humans. The virus continually changes its sequence, modifying individual sites by a process called mutation. A particular mutation in its 627 domain has long been known to enable adaptation, and this study finally provides a molecular explanation for this phenomenon. The researchers show how this mutation, that forms a complete positively charged surface, allows the polymerase to interact with human ANP32A, in the form of less specific but more numerous, weaker interactions.
This study provides a molecular framework for understanding the differential binding modes underlying the restriction of influenza polymerase by ANP32A in certain species and will allow the identification of new targets for influenza inhibition.

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A. Zarco AC, Kalayil S, Maurin D, Salvi N, Delaforge E, Milles S, Jensen MR, Hart DJ, Cusack S, Blackledge M. Nature Communications ; 2020 Jul 21 ;11(1):3656

Contact : Martin Blackledge

A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction

A new microfabrication process, developed by the IBS Synchrotron group, in collaboration with LOF Bordeaux, enables the integration of regenerated cellulose dialysis membranes between two layers of the microchip, therefore proposing a robust and inexpensive way to fabricate polyvalent microchips.
They cover the whole process, from the crystal growth on chip with the desired properties (size, number, crystal quality) by the micro-dialysis method, to the in situ X-ray diffraction experiments on several isomorphic crystals.
This approach does not require any handling of the protein crystals prior to the diffraction experiment and the collection of crystallographic data at room temperature allow the resolution of three-dimensional structure of proteins, with a much lower background noise than that generated by commercial crystallization plates used for diffraction experiments under the same experimental conditions.
The results presented here allow serial crystallography experiments on synchrotrons and X-ray lasers under dynamically controllable sample conditions to be observed using the developed microchips.

A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction. Junius N, Jaho S, Sallaz-Damaz Y, Borel F, Salmon JB, Budayova-Spano M. Lab on a Chip ; 20(2):296-310

Contact : Monika Budayova-Spano

How a bacterium manages to go unnoticed and increase its virulence

The capsule is the dominant Streptococcus pneumoniae virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, IBs researchers, in collaboration with researchers from UCL Medical School, St George’s University of London and Leceister University, describe an unexpected relationship between mutation of adcAII, which encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of adcAII resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased S. pneumoniae virulence in mouse models of infection. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of ΔadcAII, further investigation of which could further characterize mechanisms of capsule regulation.

Deletion of the zinc transporter lipoprotein AdcAII causes hyperencapsulation of Streptococcus pneumoniae associated with distinct alleles of the Type I restriction modification system. Claire Durmort, Giuseppe Ercoli, Elisa Ramos-Sevillano, Suneeta Chimalapati, Richard D. Haigh, Megan De Ste Croix, Katherine Gould, Jason Hinds, Yann Guerardel, Thierry Vernet, Marco Oggioni, and Jeremy S Brown. Mbio ; 11, 2 e00445-20

Contact : Claire Durmort

Fluorescent proteins dance in the dark

The possibility of photoconvertible fluorescent proteins (PCFPs) to convert from a green- to a red-emitting state is widely used in single-molecule localization microscopy. However, single-molecule imaging is highly disturbed by ‘blinking’ (ie transient loss of fluorescence), which is a consequence of individual PCFPs visiting « dark states ». Blinking has generally been described in the photoconverted red state, but in this paper, we focused on green-state blinking.
In a collaboration between IBS/DYNAMOP, KU Leuven (Belgium) and University Paris-Saclay, we studied dark-state formation in green mEos4b, a popular PCFP, by using UV-VIS and Raman spectroscopy, X-ray crystallography and MD simulations. We discovered that formation of one main dark state originates from cis-trans isomerization of the chromophore, similarly to what happens in so-called reversibly switchable fluorescent proteins (RSFPs). However, we found that mEos4b cannot be completely switched off (ie its « switching contrast » is low), because its chromophore wiggles a lot in the dark state and easily returns to the on fluorescent state. By comparing the number of H-bonding interactions maintained by the chromophore in different fluorescent proteins in their on- and off-states, we could suggest that the switching contrast in RSFPs is tuned by the relative strength by which the chromophore is anchored to the protein matrix in the dark compared to the bright state.

Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State. Elke De Zitter, Jacqueline Ridard, Daniel Thedie, Virgile Adam, Bernard Levy, Martin Byrdin, Guillaume Gotthard, Luc Van Meervelt, Peter Dedecker, Isabelle Demachy, Dominique Bourgeois. Journal of the American Chemical Society, 2020, ja-2020-01880m (10.1021/jacs.0c01880)

Contact : Dominique Bourgeois

Structural basis for the catalytic activities of the multifunctional enzyme quinolinate synthase

The biological cofactor nicotinamide adenine dinucleotide (NAD) is involved in many central metabolic reactions involving the transfer of one proton and two electrons. Depending of the organism, its precursor quinolinic acid (QA) is synthesized from tryptophan (like in humans) or from the condensation of dihydroxyacetone phosphate and iminoaspartate (in bacteria). The latter reaction, which is probably the oldest way to make QA, is catalyzed by the highly versatile enzyme NadA. Indeed, besides the condensation reaction, this protein catalyzes a dephosphorylation, an isomerization, a cyclization and two dehydration steps. The essential [4Fe-4S] cluster is bound at one end of the active site and is connected to the protein surface through a tunnel that can be open or closed depending on the nature (or absence) of the bound ligand.

Several crystal structures and the corresponding X-ray diffraction data for complexes of NadA with inhibitors, substrate analogs, at least one substrate (DHAP), product and potential intermediates of QA synthesis, are available from the Protein Data Bank. Based on a systematic analysis of these structures a coherent and comprehensive view of NadA catalysis is proposed in Coordination Chemistry Reviews by researchers from the Metallproteins group.

Structural basis for the catalytic activities of the multifunctional enzyme quinolinate synthase. Volbeda A, Fontecilla-Camps JC. Coordination Chemistry Reviews, Volume 417, 15 August 2020, 213370. https://doi.org/10.1016/j.ccr.2020.213370

Contact : Juan Fontecilla-Camps, Anne Volbeda

Structural insights into the mechanism of the radical SAM carbide synthase NifB, a key nitrogenase cofactor maturating enzyme

Nitrogenase, a key player in the global nitrogen cycle, catalyzes the reduction of atmospheric N2 to 2 NH3 at ambient temperature and normal pressure. Its active site, also designated FeMo-cofactor, corresponds to a [MoFe7S9C-(R)-homocitrate] species, whose biosynthesis and insertion requires the action of over a dozen accessory proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical S-adenosyl-L-methionine protein NifB plays an essential role, concomitantly inserting a carbide ion and fusing two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co.
Researchers from the Matalloproteins group determined the long-sought X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 Å resolution in a state pending the binding of one [Fe4S4] cluster substrate. The structure reveals a unique ligand binding mode for the K1 cluster, involving two cysteine residues in addition to a histidine and glutamate. A loop holding two conserved residues is inserted in the active site, likely protecting the already present [Fe4S4] clusters and regulating the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein.

Structural insights into the mechanism of the radical SAM carbide synthase NifB, a key nitrogenase cofactor maturating enzyme. Sosa Fajardo A, Legrand P, Payá-Tormo L, Martin L, Pellicer Martinez MT, Echavarri-Erasun C, Vernède X, Rubio LM, Nicolet Y. J Am Chem Soc. 2020 May 30 ;142(25):11006-11012.
doi : 10.1021/jacs.0c02243.

Contact : Yvain Nicolet

Molecular mechanism of light-driven sodium pumping

Light-driven Na+ pump KR2 is microbial rhodopsin found in marine bacteria in 2013. Its unique ability to actively transport Na+, but not K+, Ca2+, and H+, makes the protein a perspective tool for optogenetics – the biotechnological method for precise and minimally-invasive optical control of the living matter. The molecular mechanism of light-driven Na+ pumping has not yet been known, as the existing 3D structures of the KR2 were limited only to its inactive, ground state.
The researchers of IBS/Membrane Transporters group, with a support from ESRF, EMBL, Research Center Juelich, Moscow Institute of Physics and Technology, and ALBA synchrotron, solved the crystal structure of the pentameric (sodium pumping state) KR2 in its active, key intermediate state. The structure revealed a transient Na+ binding site inside the rhodopsin. Structure-based molecular dynamics simulations predicted Na+ pathway through the protein, which was further validated by the mutational analysis. The findings allowed the researchers to show that active light-driven Na+ transport proceeds via a combination of relay mechanism and passive diffusion of the ions through the polar cavities inside KR2.
It is shown that the mechanism of non proton cation transport is principally different from Grotthuss mechamism of proton transport in proton transporters like classical bacteriorhodopsin rhodopsin. The description of the Na+ binding site and the ion pathway in the protein facilitates the rational engineering of the enhanced KR2-based optogenetic tools.

Molecular mechanism of light-driven sodium pumping. Kovalev K, Astashkin R, Gushchin I, Orekhov P, Volkov D, Zinovev E, Marin E, Rulev M, Alekseev A, Royant A, Carpentier P, Vaganova S, Zabelskii D, Baeken C, Sergeev I, Balandin T, Bourenkov G, Carpena X, Boer R, Maliar N, Borshchevskiy V, Büldt G, Bamberg E, Gordeliy V. Nature Communications 2020, 11:2137

Contact : Valentin Gordeliy

COVID19 : IBS info

The IBS re-opens step by step from 25 May

Following the french government’s announcement to lift coronavirus lockdown, the gradual reopening of the IBS is being organised in accordance with all safety measures and regulations recommended by our operating agencies. The IBS re-opens step by step from 25 May and should reach a level of about 50% physical presence on site after 4 weeks. It implies that part of your contacts are still teleworking and may be reached by email (to know more).

IBS mobilized to fighting the SARS-CoV-2 Coronavirus pandemic

In order to contribute to the international scientific effort on Covid-19, IBS scientists have launched research programmes related to the SARS-CoV-2 Coronavirus (to know more).

Seminars & events

All scientific events until end of July are either cancelled or postponed (to know more).

Internships

The internships due to start after 17 March have been cancelled, and according to the UGA indications, transformed into bibliographic internships.

Looking at long non-coding RNAs from a 3D structural perspective

Noncoding RNAs accomplish a remarkable variety of biological functions. From the regulation of gene expression to translation and even the protection of genomes from foreign nucleic acids. Among them, long non-coding RNA (lncRNA) are regulating RNAs of large size (> 1000 nucleotides) that are involded in disease prevention but their function as well as three-dimensional structures remain poorly characterized. A recent study has demonstrated the role of local structural elements of the maternally-expressed gene 3 (MEG3) that potentiates protein p53, a key transcription factor controlling cell proliferation, whose role is to arrest the growth of unhealthy cells before they degenerate into cancerous tissues (Uroda T, Anastasakou E, Rossi A, Teulon J-M, Pellequer J-L, Annibale P, Pessey O, Inga A, Chillon I and Marcia M (2019) Conserved pseudoknots in lncRNA MEG3 are essential for stimulation of the p53 pathway. Mol. Cell 75 : 1-14. DOI:10.1016/j.molcel.2019.07.025).

In a collaborative work between the European Molecular Biology Laboratory (EMBL) in Grenoble with colleagues at the Max Delbruck Center in Berlin and the Institut de Biologie Structurale (CEA/CNRS/UGA) in Grenoble, researchers used complementary well established structural biology techniques to characterize the shape of lncRNA : atomic force microscopy, which identified captured individual RNA particles and inferred their size and compactness ; and small angle X-ray scattering, which characterized the RNA 3D shape in solution. The description of this new approach has been presented in a recent issue of Nature Protocols. This protocol is likely applicable to other long RNA molecules such as the untranslated RNAs (UTR) present in large RNA genome of viruses (details).

Visualizing the functional 3D shape and topography of long non coding RNAs by single-particle atomic force microscopy and in solution hydrodynamic techniques. Uroda T, Chillon I, Annibale P, Teulon JM, Pessey O, Karuppasamy M, Pellequer JL, Marcia M. Nature Protocols ; doi : 10.1038/s41596-020-0323-7

Tribute to Jean-Luc Ferrer

The IBS pays tribute to Jean-Luc Ferrer who passed away on April 21st, 2020 in his 55th year, after a long battle against the illness. He was a real pillar of the French CRG lines at ESRF, and an internationally recognized scientist in the field of structural biology.

After graduating from the Ecole Centrale Paris as an engineer in 1987, Jean-Luc Ferrer performed a DEA in Physical Chemistry at the University Paris VI, and in 1990 he obtained his PhD in Physics, which was carried out at the Bruyères-le-Châtel CEA center and focused on the dynamics of a free electron laser spectrum. Following this, Jean-Luc was recruited by the CEA Life Sciences Department at the Grenoble CEA center and joined the Laboratoire de Cristallographie et Cristallogenèse des Protéines (LCCP) directed by Juan Fontecilla-Camps and created by Michel Suscillon as part of the "Protéine 2000" program. This initiative had been taken following the decision to build the European Synchrotron (ESRF) in Grenoble. At the LCCP, Jean-Luc joined Michel Roth’s team, whose mission was to build the French BM02-D2AM beamline at the ESRF, dedicated for half of its time to protein crystallography. In 1992, the LCCP joined the IBS newly created by Jean-Pierre Ebel. A few years later, Jean-Luc also contributed to the design and construction of the new French CRG beamline dedicated to protein crystallography : BM30A-FIP.

From the very beginning, Jean-Luc Ferrer showed great interest and skills for technical and scientific aspects, sometimes very complex, of beamline construction and protein structure determination. For the latter, he initiated a very fruitful and long-lasting collaboration with Prof. Joseph P. Noel (La Jolla, USA) on the structural biology of phenylpropanoid synthesis in plants. He established collaborations with numerous French and international teams, but also developed his own research themes (consult J-L Ferrer list of publications).

After Michel Roth’s retirement in 2001, Jean-Luc became responsible for the BM30A-FIP beamline. In this position, he always showed great creativity, determination and dynamism to highlight the assets of synchrotron radiation for protein crystallography. He was a pioneer in the automated analysis of diffraction data. In addition, he anticipated the automation of crystallography beamlines and helped develop the first sample loader based on a robotic arm, the CATS robot. Later, he had the idea to use the robot arm directly as a goniometer (the G-ROB robot), which allowed for the first time to diffract crystals in their crystallization plate, paving the way to ’in situ’ crystallography, crucial for the screening of ligands in the context of drug design. He ensured the technology transfer of these various innovations by creating the start-up company NatX-ray in Grenoble and San Diego. As a national platform, the FIP beamline has become over the years an essential equipment for the French and international protein crystallography communities.

Jean-Luc Ferrer has headed the Synchrotron Group (GSY) at the IBS since its creation in 2011. As part of the ESRF-EBS program, which aims to obtain a new, extremely bright X-ray source, he sought and obtained funding for the FIP beamline reconstruction and renovation project in order to provide the best service to the community. He succeeded in driving this beamline project to completion with the launch of BM07-FIP2 next fall when ESRF reopens.

Jean-Luc was a brilliant scientist, but he was also a simple and caring person, always in a good mood. Although reserved, he appreciated social events and was always up for an evening with friends. California was a place in the world where he liked to go and recharge his batteries, close to his family. He especially loved amusement parks, where he always chose the tallest rides. His kindness was sincere and created rare moments.

Jean-Luc Ferrer maintained full professional commitment in spite of his illness, which deserves our greatest respect. His work to develop French structural biology and bring it to the highest international standards will leave a long-lasting impression on our community. The IBS as a whole salute the memory of this brilliant, yet modest, scientist and extend their sincere condolences to his family and loved ones.

Bioinsecticides : does biological mean safe ?

How to determine if an insecticide is environmentally safe ? This is the question addressed by the Canadian TV (RadioCanada) as part of the show “The Green Week” in a documentary in which Guillaume Tetreau has been interviewed for its expertise on this topic. This documentary (in French), entitled « Le Bti, un larvicide inoffensif ? » and broadcasted saturday 18th April 2020, is available in replay on RadioCanada website (https://ici.radio-canada.ca/tele/la-semaine-verte/site/episodes/461286/bti-larvicide-insecticide).

Bti is a bioinsecticide – which means that it is produced by a living organism, the bacterium Bacillus thuringiensis subsp. israelensis. In the light of its apparent safety for non-target organisms, Bti has progressively replaced chemical insecticides that were historically used for mosquito control. In Europe, it has become in a decade the only insecticide authorized for mosquito control at the larval stage. In the context of the worldwide biodiversity crisis, the hegemony of Bti is worrying and a growing number of study question its status of a “safe” insecticide. This documentary aims at compiling the current knowledge on Bti impact on the environment and the ecosystems, notably enlightening available alternatives implemented in some countries.

This reportage echoes a review article recently published in the journal Science of the Total Environment, dealing with the environmental and socioeconomic aspects of mosquito control using Bti :

Environmental and socioeconomic effects of mosquito control in Europe using the biocide Bacillus thuringiensis subsp. israelensis (Bti). Brühl CA, Després L, Frör O, Patil CD, Poulin B, Tetreau G, Allgeier S. Sci Total Environ. 2020 Mar 21:137800. doi : 10.1016/j.scitotenv.2020.137800

A tightly controlled radical-based chemistry !

Radical S-adenosyl-L-methionine (SAM) enzymes belong to a vast family of catalysts. They use the one electron reduction of a [Fe4S4] cluster to cleave SAM, producing a highly reactive 5´-deoxyadenosyl radical species. The latter in turn triggers a wide variety of radical-based reactions on substrates as different as small organic molecules, proteins, DNA or RNA. The challenging reactions they catalyse makes them very promising catalysts for diverse biotechnological applications. However, the high-energy intermediates involved require fine-control of the chemistry by the protein matrix. Understanding their control mechanism is a pre-requisite for a broader use of these enzymes as synthetic tools. In this review are presented some of the latest developments in the field, focusing on the structure-function relationship of a few examples for which three-dimensional structures, in vitro and spectroscopic data as well as theoretical calculations are available, to better describe the close interaction between the chemistry performed and the tight control of the protein matrix.

Structure-function of radical SAM enzymes ; from mechanism to biotechnological applications. Nicolet Y. Nature Catalysis ; 3 , 337–350

What role can Structural Biology play in the fight against Covid-19 ?

Joanna Timmins, researcher in the Viral Infection and Cancer group at the IBS, sheds light on the role of Structural Biology in the fight against Covid-19.
Her contribution, accessible to anyone curious about science, is available on the website of the European Crystallographic Association. Please find it here.

Watching Measles virus factories at work in liquid droplets

Many viruses are known to form cellular compartments, also called viral factories. Paramyxoviruses, including measles virus, colocalize their proteomic and genomic material in puncta in infected cells. Researchers from FDP, VRM and IRPAS groups at IBS demonstrate that purified nucleo- (N) and phospho- (P) proteins of Measles virus form liquid-like membraneless organelles upon mixing in vitro. They identify weak interactions involving intrinsically disordered domains of N and P that are implicated in this process, one of which is essential for phase separation. Fluorescence allows them to follow the modulation of the dynamics of N and P upon droplet formation while NMR is used to investigate the thermodynamics of this process. RNA colocalizes to droplets, where it triggers assembly of N protomers into nucleocapsid-like particles that encapsidate the RNA. The rate of encapsidation within droplets is enhanced compared to the dilute phase, revealing one of the roles of liquid-liquid phase separation in Measles virus replication. This study allows us to observe viral factories in action for the first time. The presence of similar puncta in numerous negative sense RNA viruses suggests that the observations made here will be of general interest in the development of antiviral strategies.

Measles Virus Nucleo- and Phosphoproteins form Liquid-like Phase-Separated Compartments that Promote Nucleocapsid Assembly. Serafima Guseva Sigrid Milles, Malene Ringkjøbing Jensen, Nicola Salvi, Jean-Phillipe Kleman, Damien Maurin, Rob W.H. Ruigrok, Martin Blackledge. Science Advances ; Vol. 6, no. 14, eaaz7095

How an electron and a proton modulate protein binding to DNA

The bacterial protein RsrR that coordinates a [2Fe-2S] cluster, regulates the expression of genes involved either directly or indirectly in cell redox equilibrium. The redox state of this iron-sulfur cluster controls RsrR binding to its site in DNA ; only the +2 oxidized form binds to the nucleic acid. In 2019, the IBS/Metallo group in collaboration with Pr. Nick Le Brun (University of East Anglia, UK) published the crystal structure of RsrR (Volbeda et al., JACS 2019) that showed an unprecedented coordination of its iron-sulfur cluster by Cys, Cys, Glu and His. The comparison of the oxidized and reduced protein structures revealed two conformations for a conserved tryptophan residue (W), called Out (in green in the figure) and In (in orange), and the concomitant displacement of a histidine (H).
In this new study, both groups, in collaboration with Dr Jean-Marie Mouesca (CEA-DRF-IRIG-DIESE-SyMMES-CAMPE), have solved the crystal structure of an RsrR-DNA complex where the three key resides (W, H and Y in black in the figure) adopt a conformation closer to the Out form. By combining chemical modification of W, site-directed mutagenesis, X-ray crystallography, quantum calculations, molecular dynamics and metadynamics simulations, they have shown that Out and In correspond to the oxidized and reduced forms, respectively. In addition, they have determined that its reduction (1 in the figure) changes the pKa of the H histidine towards more basic values ; the resulting doubly-protonated H (2) leads to the significant displacement of W, H and Y (3) ; the dipole moment of W responds to electrostatic changes caused by the reduction. Finally, the In form, thus produced, dissociates from DNA (4).
This study paves the way for the understanding of the mechanism by which the RsrR protein regulates the universal NAD cofactor synthesis and function. This cofactor is involved in photosynthesis, ATP production and cell respiration.

Electron and Proton Transfers Modulate DNA Binding by the Transcription Regulator RsrR. Crack JC, Amara P, Volbeda A, Mouesca JM, Rohac R, Pellicer Martinez MT, Huang CY, Gigarel O, Rinaldi C, Le Brun NE, Fontecilla-Camps JC. J Am Chem Soc, 142(11):5104-5116 (2020).

Structural and functional insights into Cyt1Aa, a naturally crystallin insecticidal protein

How can mosquito populations be reduced without impacting the environment or inducing resistance ? This double challenge is met by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti), which produces, in the form of nanocrystals, four toxins that specifically target mosquito larvae, thereby preventing the transmission of serious diseases such as malaria, dengue fever or chikungunya. Among the four toxins produced by Bti, researchers from a consortium of 11 laboratories led by the Institute of Structural Biology focused on the Cyt1Aa toxin, thanks to which Bti escapes the resistance of mosquitoes. By combining several structural biology approaches (femtosecond serial crystallography at an X-ray free electron laser (XFEL) and complementary biochemical, biophysical and toxicological methods in combination with mutagenisis), they elucidate the full Cyt1Aa bioactivation cascade, from in vivo crystallization to its toxic activity in the target insect. This study, published on March 2, 2020, in the journal Nature Communications, pave the way for a rational tailoring of its properties to human needs.

Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade. Tetreau G, Banneville AS, Andreeva EA, Brewster AS, Hunter MS, Sierra RG, Teulon JM, Young ID, Burke N, Gruenewald TA, Beaudouin J, Snigireva I, Fernandez-Luna MT, Burt A, Park HW, Signor L, Bafna JA, Sadir R, Fenel D, Boeri-Erba E, Rosenthal M, Coquelle N, Burghammer M, Cascio D, Sawaya MR, Winterhalter M, Gratton E, Gutsch I, Federici B, Pellequer JL, Sauter NK, Colletier JP. Nature Communications ; 11, 1153

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures

X-ray crystallography is the most prolific method to determine the structure of biological macromolecules – i.e. proteins, DNA, RNA and complexes thereof. It is limited by the strong damage inflicted to biological molecules by exposure to X-rays. To mitigate damage progression, crystallographic data collection has for the past decades been carried out mostly at cryogenic temperatures, yet at the risk of blocking conformational heterogeneity that can be central to biological function. The advent of serial crystallography – whereby each crystal is only exposed once, enabling distribution of the dose over a myriad of crystals – has yet allowed crystallographic experiments at room temperature to become more and more frequent, with the long-term promise of permitting time-resolved experiments on virtually all crystalline systems. The pre-requisite is, however, to have determined the maximal X-ray dose that can be safely deposited onto a crystal at room-temperature without fear of compromising the biological information. Using a new approach to raster-scanning serial crystallography, and taking advantage of the availability at ESRF-ID13 of both a highly-brilliant micro-focused X-ray beam and a latest generation detector, researchers at the IBS, the ESRF and the ILL, in collaboration with colleagues from the Universities of Oxford, San Francisco and Notre Dame, have been able to determine this dose limit and to visualize specific damage caused by X irradiation to biological molecules at room-temperature. This dose limit will serve as a yardstick for future room-temperature serial crystallography experiments to be performed at 4th generation synchrotrons. ESRF-EBS is the first of these.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures. Eugenio de la Mora, Nicolas Coquelle, Charles S. Bury, Martin Rosenthal, James M. Holton, Ian Carmichael, Elspeth F. Garman, Manfred Burghammer, Jacques-Philippe Colletier, and Martin Weik . PNAS ; doi.org/10.1073/pnas.1821522117

Photoswitching mechanism of a fluorescent protein revealed by time-resolved serial femtosecond crystallography and transient absorption spectroscopy

Photoswitchable fluorescent proteins are used as molecular markers in super-resolution light microscopy that images life biological cells at a resolution of a few tens of nanometers. These proteins can be reversibly toggled between a non-fluorescent (off) state and a fluorescent (on) state by irradiation with light at specific wavelengths. Photoswitching between on and off states involves ultra-fast excited-state processes that have been recently characterized structurally. Conformational changes on the slower time scale, however, have remained elusive, hampering a comprehensive description of the photoswitching mechanism at the molecular level. Using time-resolved serial crystallography at the X-ray free electron laser (XFEL) at SACLA, Japan, in combination with transient absorption spectroscopy, researchers from the IBS, the ILL and the Universities of Lille and Rennes, in collaboration with colleagues from the Max-Planck Institutes in Heidelberg and Göttingen, Germany, have now clarified the photoswitching mechanism of rsEGFP2 by determining the three-dimensional structure of a key photo-intermediate (i.e. the one populated 10 nanoseconds after photoexcitation of the off state, see figure). This study clarifies the order of events during the off-to-on photoswitching and is anticipated to facilitate rational improvement of reversibly photoswitchable fluorescent proteins for applications in super-resolution light microscopy of biological cells.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved serial femtosecond crystallography and transient absorption spectroscopy. Woodhouse J, Nass-Kovacs G, Coquelle N, Uriarte LM, Adam V, Barends TRM, Byrdin M,. de la Mora E, Doak RB, Feliks M, Field M, Fieschi F, Guillon V, Jakobs S, Joti Y, Macheboeuf P, Motomura K, Nass K, Owada S, Roome CM, Ruckebusch C, Schirò G, Shoeman RL, Thepaut M, Togashi T, Tono K, Yabashi M, Cammarata M, Foucar L, Bourgeois D, Sliwa M, Colletier JP, Schlichting I, Weik M. Nature Communications ; 11, 741