Institut de Biologie StructuraleGrenoble / France


The main target of HIV studied from every angle

By closely studying CCR5, one of the entry points of HIV into cells, researchers from Inserm, the Institut Pasteur, the University of Toulouse III - Paul Sabatier, the CNRS and IBS, demonstrate that its morphology determines the propensity of the virus to infect the body. This work supported by the ANRS and published in the Plos Pathogens journal is a new step towards understanding the role of CCR5 in HIV infection and as a target for blocking the entry of the virus into cells (details).

CCR5 structural plasticity shapes HIV-1 phenotypic properties. Colin P, Zhou Z, Staropoli I, Garcia-Perez J, Gasser R, Armani-Tourret M, Benureau Y, Gonzalez N, Jin J, Connell BJ, Raymond S, Delobel P, Izopet J, Lortat-Jacob H, Alcami J, Arenzana-Seisdedos F, Brelot A, Lagane B. PLoS Pathog. 2018 Dec 6;14(12):e1007432. doi: 10.1371/journal.ppat.1007432. eCollection 2018 Dec.

Innate immune protein C1q aggregates nanodiamonds and modifies macrophage response

Nanodiamonds (NDs) have many potential biomedical applications, such as cancer therapy, deep brain stimulation, and dentistry. In collaboration with the Service de Chimie Bioorganique et de Marquage (SCBM, CEA, Université Paris-Saclay), IBS researchers have shown that, even though C1q does not trigger the immune complement system cascade, its interaction modifies phagocytosis and cytokine response to NDs and may interfere with the multiple physiological and pathological processes that involve C1q.

Recognition protein C1q of innate immunity agglutinates nanodiamonds without activating complement. Belime A, Thielens NM, Gravel E, Frachet P, Ancelet S, Tacnet P, Caneiro C, Chuprin J, Gaboriaud C, Schoehn G, Doris E, Ling WL. Nanomedicine-Nanotechnology, Biology and Medicine; doi: 10.1016/j.nano.2018.09.009

Chromatin without a twist

Our genetic information is encoded by DNA, which is packaged in the cell nucleus in the form of chromatin. The basic building block of chromatin is the nucleosome, formed by the wrapping of DNA around a core of basic proteins called histones. Nucleosomes pack together to form a nucleosomal array, whose structure is highly dynamic and whose conformation plays a key role in gene expression. Notably, the formation of a compact 30-nm fiber is associated with the inactivation of gene expression. However, how chromatin undergoes a change in conformation remains poorly understood. In collaboration with researchers from Grenoble, Lyon and Strasbourg, IBS researchers investigated the structure of a 6-nucleosome array using a combination of structural, biophysical and biochemical approaches. The 6-nucleosome array forms a surprisingly flat structure, whose nucleosome density is only half that of the 30 nm chromatin fiber. Moreover, a minor change in ionic conditions induces the array to adopt a more compact, twisted conformation that corresponds to that of the 30 nm fiber. These results reveal how a minor change in local environment, generated for example by the post-translational modification of histones, can induce a radical change in chromatin conformation, providing insights into the structural plasticity of chromatin that is central to the regulation of gene expression.

Structure of an H1-bound 6-nucleosome array reveals an untwisted two-start chromatin fiber conformation. Isabel Garcia-Saez, Hervé Menoni, Ramachandran Boopathi, Manu S. Shukla, Lama Soueidan, Marjolaine Noirclerc-Savoye, Aline Le Roy, Dimitrios A. Skoufias, Jan Bednar, Ali Hamiche, Dimitar Angelov, Carlo Petosa, Stefan Dimitrov. Molecular Cell, doi: 10.1016/j.molcel.2018.09.027.

A key step in mitochondrial biogenesis revealed by structural biology

Mitochondria synthesize the adenosine triphosphate (ATP) molecule, which is able to transport chemical energy within cells. The amount of ATP transported daily through the mitochondrial membranes to supply our cells corresponds approximately to our body weight. This transport of mitochondrial ATP is carried out by membrane proteins, produced themselves outside the mitochondria, and which have to be inserted into the membrane where they « work ». Because these membrane proteins are insoluble in cells, their transport is extremely difficult : they may aggregate, which would be a great danger for the cell. Cells have therefore developed carriers of these membrane proteins known as "chaperones". The essential chaperones escort membrane proteins through the intermembrane space, but the structural and mechanistic details remain elusive.
Scientists from the IBS, in collaboration with EMBL, Freiburg and Tübingen Universities in Germany and University of Copenhagen in Denmark, used an integrated structural biology approach to reveal the functional principle of TIM chaperones : multiple clamp-like binding sites hold the mitochondrial membrane proteins before releasing them to their final destination.
These findings, published in Cell on November 15, may be helpful to fight diseases caused by the accumulation of protein molecules, in particular the Mohr-Tranebjærg syndrome, a neurological disorder of deafness and dystonia, caused by a dysfunction of these chaperones. Details can be found on Science Direct and within Freiburg press release.

Structural Basis of Membrane Protein Chaperoning Through the Mitochondrial Intermembrane Space. Katharina Weinhäupl, Caroline Lindau, Audrey Hessel, Yong Wang, Conny Schütze, Tobias Jores, Laura Melchionda, Birgit Schönfisch, Hubert Kalbacher, Beate Bersch, Doron Rapaport, Martha Brennich, Kresten Lindorff-Larsen, Nils Wiedemann, Paul Schanda. Cell 175, 1365–1379

Cécile Morlot is the recipient of the CNRS bronze medal

Cécile Morlot (IBS/Pneumococcus group) is the recipient of a bronze medal of the CNRS 2018. This distinction rewards an on-going and fruitful research activity, which makes him/her a specialist with talent within a particular research field.

During her thesis at the Institute for Structural Biology (IBS) in the group of Thierry Vernet, Cécile developed a fluorescent labelling method to localize, using optical microscopy, proteins in charge of cell division in an important human pathogen : Streptococcus pneumoniae (the pneumococcus). For the first time, these large protein assemblies were visible in the cell at a resolution of a few hundred nanometers. In parallel, she solved the crystallographic structure of one component of these complexes to constrain the model proposed for S. pneumoniae division.
Following her Ph.D. (2003), she completes her training in crystallography as a postdoctoral fellow in the group of Stephen Cusack (EMBL, Grenoble). Next, she enlarges her competences in microbiology during a second postdoctoral internship in the group of David Rudner (Harvard Medical School, Boston), during which she studies two protein complexes involved in spore development in Bacillus subtilis.

She is recruited by the CNRS in 2010 and joins the Pneumococcus group at the IBS to continue her research activities on bacterial morphogenesis and division, using complementary techniques in structural and cellular biology. Her recruitment coincides with the emergence of super-resolution fluorescence microscopy techniques, which allow connecting protein and cellular scales. She decides to develop the use of these techniques in the pneumococcus in collaboration with biophysicists from the IBS (Dominique Bourgeois and Virgile Adam, Pixel team) and a chemist from the University Grenoble Alpes (Yung-Sing Wong, Department of Molecular Pharmacochemistry). The developed methods, based on the localization of single molecules and on "click chemistry", now allow her to image the assembly and activity of protein machineries in charge of cell division at a resolution of about ten nanometers. Because it reveals molecular details that are inaccessible at low resolution, her work in structural biology and cell imaging helps understanding how bacteria acquire their shape and proliferate. This fundamental knowledge is pertinent for the discovery of new antibiotics and for the comprehension of associated resistance mechanisms.

New insights into 5-HT3, a serotonin receptor

This result, published in Nature, describes the activation cycle of the 5-HT3 receptor, belonging to the family of serotonin receptors. These receptors influence various biological and neurological processes such as anxiety, appetite, mood and nausea. 5-HT3 receptors are the main target of anti-emetic drugs widely used to alleviate the side effect of chemotherapies.

Scientists from the IBS, the Institut Pasteur, the University of Lorraine, the University of Copenhagen, Danemark, the University of Illinois, US, and the biotech company Theranyx, solved the structure of the 5-HT3 receptor in four different conformations. These four snapshots taken at different steps of the activation cycle of the receptor allow to describe its molecular mechanism. They also provide structural knowledge for pharmacology, revealing details of the serotonin and drug binding site, and may therefore help the development of more efficient anti-emetics (see ESRF press release).

Conformational transitions of the serotonin 5-HT3 receptor. Lucie Polovinkin​, Ghérici Hassaine, Jonathan Perot, Emmanuelle Neumann, Anders A. Jensen, Solène Lefebvre​, Pierre-Jean Corringer, Jacques Neyton, Christophe Chipot​, Francois Dehez, Guy Schoehn ​& Hugues Nury. Nature 563(7730):275-279

Structure of an enzyme complex essential for the metabolism of the bacterial wall of important pathogens

The universality of peptidoglycan in bacteria underlies the broad spectrum of many successful antibiotics. However, in our times of widespread resistance, the diversity of peptidoglycan modifications offers a variety of new antibacterials targets. In some Gram-positive species such as Streptococcus pneumoniae, Staphylococcus aureus, or Mycobacterium tuberculosis, the second residue of the peptidoglycan precursor, D-glutamate, is amidated into iso-D-glutamine by the essential amidotransferase MurT/GatD complex. Here, IBS/PG researchers in collaboration with the Norwegian University of Life Sciences, Newcastle University, Universidade Nova de Lisboa, Utrecht University and Université Paris Descartes, present the structure of this complex at 3.0 Å resolution. MurT has central and C-terminal domains similar to Mur ligases with a cysteine-rich insertion, which probably binds zinc, contributing to the interface with GatD. The mechanism of amidation by MurT is likely similar to the condensation catalyzed by Mur ligases. GatD is a glutaminase providing ammonia that is likely channeled to the MurT active site through a cavity network. The structure and assay presented here constitute a knowledge base for future drug development studies.

Structure of the essential peptidoglycan amidotransferase MurT/GatD complex from Streptococcus pneumoniae. Morlot C, Straume D, Peters K, Hegnar OA, Simon N, Villard AM, Contreras-Martel C, Leisico F, Breukink E, Gravier-Pelletier C, Le Corre L, Vollmer W, Pietrancosta N, Håvarstein LS, Zapun A. Nature Communcations;9(1):3180

Structural investigation of a chaperonin in action

Many fundamental cellular functions are performed by large protein assemblies. This applies in particular to protein quality control, the process by which cells ensure the recycling or correct folding of their main constituents, in order to avoid the accumulation of poorly folded proteins, aggregates or fibrils. These large machineries - chaperones, proteases and peptidases - are complex and dynamic. The studies of such biological machines present a significant challenge, due to the very size of these particles, the complexity of their biological substrates and the structural rearrangements involved. IBS researchers have implemented an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, they provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins. The results, published in Sciences Advances on September 19, 2018, reveal how the functional cycle of this complex assembly is regulated. This approach opens up new perspectives for directly studying the structures and mechanisms of various biological machines in action.

Structural Investigation of a Chaperonin in Action Reveals How Nucleotide Binding Regulates the Functional Cycle. Mas G, Guan J-Y, Crublet E, Colas Debled E, Moriscot C, Gans P, Schoehn G, Macek P, Schanda P, Boisbouvier J. Science Advances 2018 September 19

Geochemical Continuity and Catalyst/Cofactor Replacement in the Emergence and Evolution of Life

Évolution de la synthèse d'adénine et uracile depuis une surface minéral jusqu'à l'ATPCurrent hypotheses about the origin of life posit that it may have started with either the replication, in a primordial soup, of genetic information-containing polymers with limited catalytic properties, (the “RNA World”), or with autocatalytic, and possibly interacting, metabolic pathways taking place on, or near, mineral surfaces. The latter possibility can be explored if a continuous geochemical, catalytically dynamic process is assumed. In this Essay it is speculated that the synthesis of the nucleic acid purine and pyrimidine bases originated on a mineral surface, which was later replaced by ATP.

Geochemical Continuity and Catalyst/Cofactor Replacement in the Emergence and Evolution of Life. Fontecilla-Camps JC. Angewandte Chemie International Edition. doi: 10.1002/anie.201808438

ERC Starting Grant 2018 for Sigrid Milles

The European Research Council (ERC) has awarded a "Starting Grant" to Sigrid Milles, ‘flexibility and dynamics of proteins’ group at the IBS, to study intrinsically disordered proteins in endocytosis by single molecule fluorescence and nuclear magnetic resonance spectroscopy.

Sigrid Milles is a CNRS researcher at the Institut de Biologie Structurale. Her project, named ‘MultiMotif’, has been selected for funding through an ERC Starting Grant over the next five years. Scientific excellence at European level is one of the main criteria for the selection of these awards which recognize innovative projects of promising researchers, who finished their doctorate 2 to 7 years ago and want to create or consolidate a research team.

After a PhD on single molecule fluorescence spectroscopy at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany (2013), Sigrid Milles joined Martin Blackledge’s group at the IBS for a postdoc in nuclear magnetic resonance (NMR) spectroscopy. During her thesis and postdoctoral studies, Sigrid studied intrinsically disordered proteins, that means proteins without a stable three-dimensional structure. She was first interested in the proteins of the nuclear pore complex, a transport channel that connects the nucleoplasm and the cytoplasm, and her work has allowed to understand how transport through the pore can at the same time be fast (milliseconds) and specific (published in Cell, 2015). More recently, Sigrid worked on the intrinsically disordered proteins of the measles virus replication machinery and she has just discovered a new interaction site between two viral proteins (published in Science Advances, 2018), which might present a new future target to treat measles infection. She got recruited by the CNRS in 2017 and aims now to combine fluorescence and NMR spectroscopy to study the intrinsically disordered proteins in endocytosis – the major transport pathway into the eukaryotic cell.

What is this project about ?
Endocytosis is responsible for the entry of molecules into the eukaryotic cell, as for example nutrients, signaling molecules and their receptors, but also pathogens. This mechanism is thus very important and relies on the small molecule clathrin (clathrin-dependent endocytosis), which forms the structural scaffold shaping the membrane and finally resulting in the uptake of a coated vesicle. However, a lot of other proteins are necessary for this highly regulated uptake process, amongst others proteins that contain long intrinsically disordered regions. These regions are interspersed with small sequence stretches, called linear motifs, which interact with different binding partners from the endocytosis machinery. Although these interactions are crucial for endocytosis, they are not very well understood due to the flexibility and dynamics of the protein sequences they are embedded in.
This ERC project aims at developing an integrated approach using single molecule fluorescence and NMR spectroscopy to study these intrinsically disordered proteins and understand the molecular mechanism by which their linear motifs regulate the process of endocytosis. Understanding the way of function of these motifs is important not only for endocytosis, but also many other biological processes that also rely on using linear motifs.

Keywords : Single molecule fluorescence, nuclear magnetic resonance, intrinsically disordered proteins, endocytosis

Amount of the award : €1.599 million for five years

First results published from the European XFEL

View into the 3.4km long tunnel of the European XFEL (©IBS/M.Weik)

X-ray free-electron lasers (XFELs) are novel X-ray sources that provide femtosecond pulses of a peak brilliance that exceeds that of synchrotron sources by nine orders of magnitude. The short duration of the pulses matches the chemical time scale of femtoseconds, allowing the investigation of the dynamics of matter in a time-resolved manner and enables the analysis of highly radiation-sensitive objects. IBS researchers participated in one of the first experiments at the newly built European XFEL in Hamburg, Germany, lead by the Max Planck Institute (MPI) for Medical Research in Heidelberg. This large-scale facility is the first producing XFEL pulses with a MHz repetition rate, to be compared with the 60 and 120 Hz rate of the XFELs at SACLA (Japan) and the LCLS (USA), respectively.
They investigated a microcrystalline preparation of jack bean proteins precipitated with acetone, a preparation described by James Sumner, who used this technique for the first crystallization of an enzyme (urease) in 1926. That work ultimately showed that enzymes are proteins and resulted in a Nobel Prize in 1946. Together with their colleagues of the MPI in Heidelberg, the Universities of Rennes and Lille and others they demonstrated that it is possible to separate the data of the three protein crystals in this mixture (urease, concanavalin A and B) and to determine the structures of the two concanavalins, using data collected at the first MHz XFEL. Furthermore, the consortium showed that under the current operating conditions of the European XFEL, data quality is independent of whether the first or subsequent pulses of the train were used for data collection, i.e. that shockwave- and other effects do not compromise data quality at MHz repetition rates.
These results, published in Nature Communications on August 28, are of interest to a growing community of scientists interested in using MHz XFELs. In a near future, XFEL technology will become available to many more scientists since the cost of these measurements will decrease greatly as the time spent to acquire the data is reduced. The techniques used in the present work and the underlying physics of operating sequential experiments at MHz rates are directly relevant to other subfields of XFEL science, from physicists interested in extreme interactions between radiation and matter, to chemists focused on ultrafast reactions, and to other scientists interested in “big data” measurements.

Megahertz data collection from protein microcrystals at an X-ray free-electron laser. Grünbein ML, Bielecki J, Gorel A, Stricker M, Bean R, Cammarata M, Dörner K, Fröhlich L, Hartmann E, Hauf S, Hilpert M, Kim Y, Kloos M, Letrun R, Messerschmidt M, Mills G, Nass Kovacs G, Ramilli M, Roome CM, Sato T, Scholz M, Sliwa M, Sztuk-Dambietz J, Weik M, Weinhausen B, Al-Qudami N, Boukhelef D, Brockhauser S, Ehsan W, Emons M, Esenov S, Fangohr H, Kaukher A, Kluyver T, Lederer M, Maia L, Manetti M, Michelat T, Münnich A, Pallas F, Palmer G, Previtali G, Raab N, Silenzi A, Szuba J, Venkatesan S, Wrona K, Zhu J, Doak RB, Shoeman RL, Foucar L, Colletier JP, Mancuso AP, Barends TRM, Stan CA, Schlichting I. Nature Communications; volume 9: 3487

Revealing molecular mechanisms that prevent measles virus replication

Info presseIBS Researchers, in collaboration with the CIRI, discovered a novel interaction between two proteins from the measles virus. This ultra-weak interaction, involving only four amino acids situated in a very flexible and dynamic protein region, is essential for measles virus replication. This newly discovered interaction constitutes a new target to treat measles infection, but also infection by other viruses from the same family, that comprises highly dangerous human pathogens. These results are published in Science Advances, on August 22nd, 2018.

Press information

An ultra-weak interaction in the intrinsically disordered replication machinery is essential for Measles virus function. S. Milles, M. R. Jensen, C. Lazert, S. Guseva, S. Ivashchenko, G. Communie, D. Maurin, D. Gerlier, R. W. H. Ruigrok, M. Blackledge. Sci. Adv. 4, eaat7778

A modified Fe-S cluster modulates [Ni-Fe] hydrogenase oxidative damage

Hydrogenases are enzymes of considerable interest for their potential biotechnological applications both as catalysts in biofuel cells and hydrogen producers. However, these applications can be greatly affected by their reactions with atmospheric oxygen. In order to better understand this problem, we have investigated a single mutation of the naturally O2-tolerant E. coli [NiFe] hydrogenase-1, which makes it O2-sensitive by changing its [4Fe-3S] cluster into a novel [4Fe-4S] cluster. Our theoretical study explains in detail the observed different redox properties of these two clusters and sheds considerable light on the biological solution to prevent O2-based deactivation.

X-ray structural, functional and computational studies of the O2-sensitive E. coli hydrogenase-1 C19G variant reveal an unusual [4Fe–4S] cluster. A. Volbeda, J. M. Mouesca, C. Darnault, M. M. Roessler, A. Parkin, F. A. Armstrong and J. C. Fontecilla-Camps. ChemComm; accepted 4th June 2018, DOI: 10.1039/c8cc02896f

C1q and MBL opsonins use a common anchor site on the CR1 receptor

Complement receptor type 1 (CR1) is a multi modular membrane receptor involved in the clearance of complement opsonized components from the blood stream. By binding targets tagged with complement-opsonins, the CR1 receptor on the surface of erythrocytes contributes to their elimination by transport to the liver, then phagocytosis by monocytes, macrophages or neutrophils.
CR1 is composed of 30 homologous complement control protein (CCP) modules and is a receptor for complement-opsonins C3b and C4b. While C3b and C4b have long been known as ligands of CR1, it is only recently that defense collagens such as mannose-binding lectin (MBL), ficolin-2, and C1q have also been shown to act as opsonins.
In this study, the IRPAS group located the attachment site of C1q and MBL to CR1 thanks to a molecular dissection strategy particularly adapted to the study of multi-modular proteins. The interaction site for both MBL and C1q is located on the same pair of modules CCP24-25 out of the 30 modules in CR1. This study contributes to enlarge knowledge on the multifunctional role of complement defense collagens and more especially on this new opsonin function that allows the transfer of foreign agents or altereded self on various receptors for clearance.

C1q and MBL interact with CR1 in the same region on CCP24-25 modules. Jacquet M, Cioci G, Fouët G, Bally I, Thielens NM, Gaboriaud C, Rossi V. Frontiers in Immunology;9, 453

How Detergent Impacts Membrane Proteins

Many cellular processes involve membrane proteins (MPs) and characterization of their structure, interactions and dynamics remains a challenge for structural biology. The difficulty is related to the need to extract these proteins from their biological membrane in order to study them. Detergents are frequently used but their physical properties differ from those of lipids and could alter the structural organization of MPs.
In this study, a family of membrane proteins, mitochondrial transporters, were analyzed in detail in a common detergent, dodecylphosphocholine (DPC). Several studies on these mitochondrial carriers in DPC had already proposed details on structure, dynamics and interactions and interpreted these observations from a biological perspective, but their biological relevance has been questionned. The NMR and MEMBRANE group studies, in collaboration with researchers at Nancy and Cambridge, combine NMR, biochemistry and MD simulation methods to resolve unambiguously this controversy. The results show a subtle balance of interactions between protein and detergent, which induces a significant disruption of the protein; the specificity of interaction with substrates is strongly impacted, and the protein samples partially unfolded conformations. A review of a number of structures obtained in DPC illustrates that these effects are relatively general, and helps clarify a debate on the impact of detergents.

1) How Detergent Impacts Membrane Proteins: Atomic-Level Views of Mitochondrial Carriers in Dodecylphosphocholine. Kurauskas V, Hessel A, Ma P, Lunetti P, Weinhäupl K, Imbert L, Brutscher B, King MS, Sounier R4, Dolce V, Kunji ERS, Capobianco L, Chipot C, Dehez F, Bersch B, Schanda P. Journal of Physical Chemistry Letters;9(5):933-938
2) Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies. Chipot C, Dehez F, Schnell JR, Zitzmann N, Pebay-Peyroula E, Catoire L, Miroux B, Kunji ERS, Veglia G, Cross TA, Schanda P. Chemical Reviews;118(7):3559-3607
3) Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant. Kurauskas V, Hessel A, Dehez F, Chipot F, Bersch B, Schanda P. Nature Structural & Molecular Biology;doi:

New light on the mevalonate bioynthetic reaction in archaea

Mevalonate is a starting material to synthesize many chemicals in industry; it is also the building block of the lipids from all archaea. Scientists at the IBS and and collaborators at the Max Planck Institute for Terrestrial Microbiology in Marburg and ENS Lyon discovered a coupling between the two enzymes responsible for the first step in mevalonate biosynthesis in archaea. This finding explain how archaea can produce mevalonate at high rate to support their growth, and can be applied in industry to optimize mevalonate production. Details

Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site. Vögeli, B., Engilberge, S., Girard, E., Riobé, F., Maury, O., Erb, T.J., Shima, S., Wagner, T. PNAS, 115(13): 3380-3385

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy

Intrinsically Disordered Proteins (IPDs) are functionally active despite lacking a well-defined three-dimensional structure. Their great flexibility allows them to easily adapt to the surface of their partners and they are able to fold during interaction. In some cases, they may even form a so-called fuzzy complex, in which the IPD does not adopt a single conformation defined on the partner’s surface, but continues to sample multiple conformations in a highly dynamic complex.
The FDP group of the IBS, in collaboration with researchers from IAB Grenoble and the ENS Paris, succeeded in obtaining an atomic resolution description of the dynamics of an IDP in complex with its partner using nuclear magnetic resonance spectroscopy. Researchers applied this approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4.. The study shows that MKK4 uses a combination of interaction modes to link to p38α, leading to a complex displaying significantly different dynamics between linked regions. The results show how IPDs can engage in very specific interactions without having a strong binding affinity.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy. Delaforge E, Kragelj J, Tengo L, Palencia A, Milles S, Bouvignies G, Salvi N, Blackledge M, Jensen MR. Journal of the American Chemical Society;140(3):1148-1158

Capture of a «phantom » state of green fluorescent proteins

Green fluorescent proteins (GFPs) are genetically encoded markers allowing the localization of down to individual proteins by optical microscopy. Their fluorophore is formed from three amino acids and is located at the center of a beta barrel that protects it from the environment. GFPs have high absorption coefficient and fluorescence yield, but only moderate photostability. They can reversibly form temporary nonfluorescent “dark” states before definitely losing fluorescence after about 100000 excitation/emission cycles.
At the IBS, the Pixel team has previously solved the structures of some of such dark states. All of them showed considerable chemical modifications which raised the question as to their irreversibility. Therefore, it was suspected that a reversible gateway state should exist the knowledge of which would allow to improve the GFP’s behavior. But little was known on this putative « phantom » state: its chemical character (triplet, radical, biradical ?), its yield (percents, permills ?), its lifetime (nano-, micro-, millisecondes ?), its reactivity (oxidative, reductive, both ?), its absorption spectrum (UV, VIS, IR ?).
Thanks to a collaboration with the team of Klaus BRETTEL at I2BC (CEA Saclay), the Pixel team of the DYNAMOP group at the IBS exposed the paradigmatic fluorescent protein EGFP to a study by transient absorption spectroscopy. This technique has previously been applied to fluorescent proteins very little. Pushing it to its resolution limits allowed the detection of the spectral signature of the « phantom » dark state and its characterization by answering all of the above questions. These findings open the way for a better understanding of the photophysical functioning of fluorescent proteins and fuel the hope for finding keys to their further optimization.

A Long-Lived Triplet State Is the Entrance Gateway to Oxidative Photochemistry in Green Fluorescent Proteins. Byrdin M, Duan C, Bourgeois D, Brettel K. Journal of the American Chemical Society;. doi: 10.1021/jacs.7b12755

How bacteria converse in floating biofilms

Biofilms are bacterial communities with high antibiotic resistance. Within biofilms, bacteria exchange information chemically - a mechanism called quorum-sensing. Researchers at the Institute de Biologie Structurale in Grenoble, the University of the Mediterranean in Marseille and the Jacobs University in Bremen have shown that the gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact,before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, they hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii, Omp-Pst1 and Omp-Pst2. The crystal structures reveal that these porins can selfassociate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication. Proteins involved in this interaction could be new targets in the fight against biofilms.

Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities. El-Khatib M, Nasrallah C, Lopes J, Tran QT, Tetreau G, Basbous H, Fenel D, Gallet B, Lethier M, Bolla JM, Pagès JM, Vivaudou M, Weik M, Winterhalter M, Colletier JP. Proceedings of the National Academy of Sciences of the United States of America. 2018 Feb 23. pii: 201714582. doi: 10.1073/pnas.1714582115.

Bacterial pathogens can reprogram target cells by influencing epigenetic factors

The type III secretion system (T3SS) is a complex nanomachine used by numerous Gram-negative bacteria to inject toxins directly into target cells. Its architecture resembles a syringe, and toxins are believe to travel through its interior. One key aspect of the T3SS is the translocon, a complex of two membrane proteins that are synthesized within the bacterial cytoplasm, transported through the interior of the needle, and subsequently inserted directly into the membrane of the eukaryotic cell, allowing toxin passage. In this work IBS scientists and their collaborators from BIG and London Imperial College showed that insertion of the translocon proteins (PopB and PopD) by the human pathogen Pseudomonas aeruginosa into target membranes engenders epigenetic modifications on histone H3 as a consequence of ion exchange through the formed pore. This thus indicates, for the first time, that the translocon acts not only as a pore, but also as a bona fide virulence factor.

Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome. Laurent Dortet, Charlotte Lombardi, François Cretin, Andréa Dessen, Alain Filloux. Nature Microbiology 2018 Feb 5. doi: 10.1038/s41564-018-0109-7

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins

The dynamic fluctuations of intrinsically disordered proteins (IDPs) define their function. Although experimental nuclear magnetic resonance (NMR) relaxation reveals the motional complexity of these highly flexible proteins, the absence of physical models describing IDP dynamics hinders their mechanistic interpretation. Combining molecular dynamics simulation and NMR, the researchers of the FDP group (Protein Dynaics and Flexibility by NMR) introduce a framework in which distinct motions are attributed to local libration, backbone dihedral angle dynamics and longer-range tumbling of one or more peptide planes. This model provides unique insight into segmental organization of dynamics in IDPs and allows them to investigate the presence and extent of the correlated motions that are essential for function.

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins. Salvi N, Abyzov A, Blackledge M. Angewandte Chemie International Edition England ;56(45):14020-14024.

Antibiotics and radical-based chemistry: the 1,2-diol dehydratase AprD4 from the inside

New sources of antibiotics are required to fight against multidrug-resistant pathogens. Natural product biosynthetic pathways are a vast source of inspiration to develop new efficient and environment-friendly chemical synthesis processes. Radical-based chemistry, using high-energy intermediates can afford difficult reactions in water. ‘Radical SAM’ enzymes control such intermediates to perform regio- and stereo-specific reactions. The crystal structure of the radical SAM 1,2-diol dehydratase AprD4, determined by the Metalloproteins unit at IBS in collaboration with the group of Pr. Qi Zhang at Fudan University (Shanghai, China), has revealed that the remarkable tridimensional arrangement at its active site, while keeping substrate-specificity, gives the radical intermediate enough freedom to adopt different conformations, in order to release a specific water molecule. This modification makes certain aminoglycoside antibiotics insensitive to the most common mechanisms of resistance toward this family of antimicrobial agents.

1,2-diol dehydration by the radical SAM enzyme AprD4 - a matter of proton circulation and substrate flexibility. Liu WQ, Amara P, Mouesca JM, Ji X, Renoux O, Martin L, Zhang C, Zhang Q, Nicolet Y. Journal of the American Chemical Society 2018 Jan 4. doi: 10.1021/jacs.7b10501. [Epub ahead of print]