Institut de Biologie StructuraleGrenoble / France


Paul Schanda ERC Starting Grant 2012

A young IBS researcher, Paul Schanda (Biomolecular NMR Spectroscopy Group) has been awarded an ERC Starting Grant* by the European Research Council (ERC) for his study of functional dynamics of proteins. The grant is worth EUR 1.49 million. Paul is the second researcher in the NMR group to obtain this prestigious grant (after Jerome Boisbouvier in 2010).

This project will investigate the link between motion and function in two challenging systems. On the one hand, the intrinsic dynamics of a membrane protein will be studied in the light of its transport activity. Membrane proteins fulfill a wide range of functions in the cell, and are a prime drug target. Yet, our current knowledge about their structure and dynamics is very limited, due to the difficulties to obtain atomic-resolution data. Importantly, for many functions, such as transport across membranes and signaling, flexibility is a key property. In this project Paul Schanda’s team will use and develop nuclear magnetic resonance techniques to provide unprecedented insight into the intimate connection between motion and function on an atomic level. As a second target, the team will also investigate another class of challenging biological objects: very large chaperones, that assist other proteins in their folding. Their sheer size makes atomic-resolution studies of chaperones at a molecular level a significant challenge. Again, a combination of solution and solid-state NMR will be used to address the function and interaction with client proteins. The main tool used for these studies will be nuclear magnetic resonance, both in solution and in the solid state. The combination of both NMR approaches will allow to go significantly beyond the capabilities of each technique alone.

The structural biologist Paul Schanda developed innovative NMR techniques during his PhD, focusing primarily at speeding up NMR data acquisition. A number of now state-of-the-art tools, have accelerated NMR by more than one order of magnitude, and allow nowadays to study short-lived protein states, such as folding intermediates in real time. For his post-doc, he switched to an emerging technique in structural biology, solid-state NMR. During his research period at ETH Zurich in B. H. Meier’s lab, he developed and applied solid-state NMR techniques for the study of protein motion in fibrillar and microcrystalline proteins. End of 2010, Paul Schanda joined IBS, where he could set up a small team funded by an ANR grant. The main goal of his team is the study of protein dynamics in challenging biological systems.

*ERC Starting Grants are awarded for a five-year period to academics leading an independent team or programme who have the potential to become world-class researchers

Pretending to be somebody else

Human and pneumococcal cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins are both ligands of human C1q protein. Terrasse R1, Tacnet-Delorme P, Moriscot C, Pérard J, Schoehn G, Vernet T, Thielens NM, Di Guilmi AM, Frachet P. J Biol Chem. 2012 Dec 14;287(51):42620-33.

Atomic-Resolution Structural Dynamics in Crystalline Proteins from NMR and Molecular Simulation

Solid-state NMR can provide atomic-resolution information about protein motions occurring on a vast range of time scales under similar conditions to those of X-ray diffraction studies and therefore offers a highly complementary approach to characterizing the dynamic fluctuations occurring in the crystal. For the first time, researchers a the IBS have compared extensive experimentally determined dynamic parameters, spin relaxation, chemical shifts, and dipolar couplings, to values calculated from a 200 ns MD simulation of protein GB1 in its crystalline form, providing new insight into the nature of structural dynamics occurring within the protein crystalline lattice. This insight will have broad impact on the interpretation of solid state NMR data in terms of protein dynamics..

Atomic-Resolution Structural Dynamics in Crystalline Proteins from NMR and Molecular Simulation. Luca Mollica, Maria Baias, Józef R. Lewandowski, Benjamin J. Wylie, Lindsay J. Sperling, Chad M. Rienstra, Lyndon Emsley, and Martin Blackledge. J. Phys. Chem. Lett., 2012, 3 (23), pp 3657–3662

New insight into the dynamical behavior of protein molecules

It has long been known that below –50 °C most enzymes suddenly loose activity and are not able to fulfill their biological role anymore. Dynamical changes appear to be responsible for this loss in function, since at the same temperature proteins become very rigid. Until now it was not clear to which extent this so-called protein dynamical transition involved all of the protein, or was dominated by surface residues and their interaction with solvent. The research led by Dr. Frans Mulder at Aarhus University, Dr. Kathleen Wood at the Australian Nuclear Science and Technology Organisation Bragg Institute and Dr. Martin Weik at the IBS and carried out in collaboration with the Deuteration Laboratory and beamline scientists at the Institut Laue-Langevin demonstrates the importance of protein solvation for flexibility and shows that the protein dynamical transition arises from a global change that affects the entire protein [1]. Understanding protein dynamics in detail will provide important insight into allosteric regulation, cellular protein signaling, and drug action.

[1] Protein Surface and Core Dynamics Show Concerted Hydration-Dependent Activation. Wood K, Gallat FX, Otten R, van Heel AJ, Lethier M, van Eijck L, Moulin M, Haertlein M, Weik M, Mulder FA. Angew Chem Int Ed doi: 10.1002/anie.201205898. [Epub ahead of print]

A fluorescent protein turns to the dark side

Upon an intense and prolonged illumination, fluorescent proteins undergo substantial transformations that irreversibly results in photobleaching, ie they lose their fluorescence properties. This phenomenon constitutes a major inconvenience for most of the fluorescence-based imaging applications, while it is also turned into an advantage for some specific techniques such as for fluorescence recovery after photobleaching. KillerRed is an engineered red fluorescent protein that reveals strong photo-cytotoxic properties, and hence used as an efficient photosensitizer in dynamic phototherapy (for instance to kill cancer cells). Essentially, it generates toxic reactive oxygen species (ROS) during the photobleaching process. The molecular mechanisms which results in the photobleaching of fluorescent proteins are complex and remain poorly characterized. In this article, we have combined X-ray crystallography with UV/Visible spectroscopy to study a green emitting mutant of KillerRed that revealed a remarkable photosensitivity. These in crystallo studies, carried out jointly at the IBS and at the ESRF, allowed us to draw a mechanistic scheme at the molecular level dissecting the laser-induced photo-conversions of the protein that end up in its photobleached state. In addition, this work provides new insights into understanding the ROS release mechanism that occurs during the photosensitizing event. It can also help to design new photo-sensitive or photo–resistant fluorescent proteins, depending on the imaging applications.

GFP-like phototransformation mechanisms in the cytotoxic fluorescent protein KillerRed unraveled by structural and spectroscopic investigations. Eve de Rosny and Philippe Carpentier. J. Am. Chem. Soc., 2012, 134 (43), pp 18015–18021

Heparan sulphate helps chemokines to direct cell migration within tissues

CXCL12 is a chemokine regulating the tissue homing and survival of circulating tissue-specific progenitors. Beyond binding to its cognate receptor CXCR4, CXCL12 also interact with heparan sulphate proteoglycans, a process that is believe to promote the formation of chemotactic gradients, which in turn determine the orientated migration and tissue recruitment of circulating cells. To test this hypothesis our collaborators at the Pasteur Institute engineered a mouse carrying a Cxcl12 gene mutation that precludes CXCL12 interactions with heparan sulphate while not affecting CXCR4-dependent cell signalling. After induced acute ischemia, a marked impaired capacity to support revascularization was observed in Cxcl12 mutant animals, demonstrating that in vivo, binding to heparan sulphate was necessary to induce progenitor cell infiltration in the ischemic tissue and to restore vascular growth

Homeostatic and tissue reparation default in mice carrying selective genetic invalidation of CXCL12/proteoglycan interactions. Rueda P., Richart A., Récalde A., Richart A., Gasse P., Vilar J., Guérin C., Lortat-Jacob H., Vieira P., Baleux F., Chretien F., Arenzana-Seisdedos F. and Silvestre J-S. Circulation 126, 1882-1895 (2012)

The very elongated structure of RecN revealed by a combined structural biology approach.

The first quasi-atomic structure of a member of the SMC (Structural maintenance of chromosomes)-like protein family, namely the RecN protein, was assembled from three crystal structures and a low-resolution envelope by scientists from the ESRF and IBS. This structure together with biochemical studies allowed us to propose a model for the role of RecN in the repair of DNA double-strand breaks.

Structural and Functional Characterization of an SMC-like Protein RecN: New Insights into Double-Strand Break Repair.
Pellegrino S, Radzimanowski J, de Sanctis D, Erba EB, McSweeney S, Timmins J.
Structure. 2012 Oct 16. pii: S0969-2126(12)00344-9. doi: 10.1016/j.str.2012.09.010.

Electronic tongue for protein analysis

Electronic tongues, a device increasingly used in the food and heath industries, consist of sensors incorporating many different receptors, often long and complex to assemble. Inspired by the mechanism by which proteins are recognized by Heparan Sulphate, the teams of Thierry Livache (DSM – INAC), Hugues Lortat-Jacob (DSV – IBS) and David Bonnaffé (Paris-Sud University) have developed a new electronic tongue system: a receptor network was made by the combinatorial self assembly of different disaccharidic building blocks. This sensor can distinguish different proteins in mixture, generating for each of them specific "2D profiles" or "3D landscapes" that facilitate their identification. This approach also offers an unprecedented and simple tool to investigate the importance of the HS overall organization (charge density and distribution along the chain) for protein binding (press release).

Continuous Evolution Profiles for Electronic Tongue Based Analysis. Hou Y., Genua M., Tada Batista D., Calemczuk R., Buhot A., Fornarelli P., Koubachi J., Bonnaffé D., Saesen E., Laguri C., Lortat-Jacob H. and Livache T. Angew Chem Int Ed Engl. 51 (41), 10394-10398 (2012).

Science Fair 2012

Atelier visualisation de cristauxFor the 2012 Science Week, the Institute organized two events on october 11 and 12 :

- IBS researchers, engineers and technicians invited secondary school pupils to discover the proteins which are essential for life. They had the opportunity to visit laboratories, accomplish scientific experiments (through RMN, electron microscopy and crystallography workshops) and get information about careers in research.

Atelier microscopie électronique Atelier RMN

- A hundred pupils were also invited to participate in one of three workshops (short experiments involving proteins and DNA). They could visit a the laboratory to discover benches, chemical products, instruments etc.

Atelier découvertes des protéines Atelier ADN bananes
Atelier protéines fluorescentes Visite labo par les CM2

Mapping the Potential Energy Landscape of Intrinsically Disordered Proteins at Amino Acid Resolution by NMR

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology.
New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins.
In this study we develop a novel approach that uses NMR data (chemical shifts or dipolar couplings) to map, for the first time, the potential energy landscape of intrinsically disordered proteins at amino acid specific resolution.
We apply these approaches to characterize the conformational behaviour of two intrinsically disordered proteins, the K18 domain from Tau protein and Ntail from Measles virus nucleoprotein.
In both cases we identify the enhanced populations of turn and helical regions in key regions for the function of these proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

Mapping the Potential Energy Landscape of Intrinsically Disordered Proteins at Amino Acid Resolution. Ozenne V, Schneider R, Yao M, Huang JR, Salmon L, Zweckstetter M, Jensen MR, Blackledge M. J Am Chem Soc. ;134(36):15138-48

A channeling mecanism is involved in the pathway of the biotin synthesis in plants

Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyse, respectively, the antepenultimate and the penultimate steps of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi) both activities are carried by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. The enzyme catalyses both DAPA-AT and DTBS reactions in vitro, and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of the 2.7 Å resolution structure of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and bring additional evidences for substrate channelling which could take place in a crevice at the surface of the enzyme. Mutation of residues observed in this crevice abolished channeling. Finally, elucidation of the structure of a plant bifunctional DTBS/DAPA-AT creates a rich molecular platform that will be useful for the design of new inhibitor families having herbicidal and fungal activities.

Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase-Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis, Cobessi, D., Dumas, R., Pautre, V., Meinguet, C., Ferrer, J.-L., Alban, C., Plant Cell;24(4):1608-25

Molecular basis of genetic diseases revealed by functional and dynamic studies of the mitochondrial ADP/ATP carrier

The mitochondrial ADP/ATP carrier plays a key role in the energetic regulation of the cell, importing ADP into the mitochondria and exporting ATP toward the cytoplasm. Severe genetic diseases have been ascribed to deficiencies of this membrane protein. In collaboration with the team of C. Chipot in Nancy (CNRS-UHP), we have characterized for the first time the molecular impact of six pathological mutations that have been pinpointed for ophthalmoplegia and myopathies. In vivo functional assays combined synergistically with molecular-dynamics simulations demonstrate that the mutations alter the transport activity of the protein by modifying its electrostatic properties, or reducing its conformational plasticity. Throwing a bridge between the pathologies and their molecular origins, these results pave the way to explore novel, well-adapted therapeutic strategies.

Impaired Transport of Nucleotides in a Mitochondrial Carrier Explains Severe Human Genetic Diseases. Stéphanie Ravaud, Axel Bidon-Chanal, Iulia Blesneac, Paul Machillot, Céline Juillan-Binard, François Dehez, Chris Chipot and Eva Pebay-Peyroula. ACS Chem. Biol., 2012, 7 (7), pp 1164–1169

Insights into the mechanism of Helicobacter pylori cytotoxin cagA internalization by host cells

Infection with Helicobacter pylori strains carrying the cytotoxin-associated antigen A (cagA) gene is associated with gastric cancer. The oncoprotein cagA is injected into gastric epithelial cells by the bacterial type IV secretion system (cag-T4SS) and exploits α5β1 integrin as a receptor for its own translocation. The cagA crystal structure reveals a modular protein composed of a central domain responsible for β1 integrin binding and a C-terminal helical domain mimicking eukaryotic cytoskeleton proteins. Characterization of the CagA/integin β1 interaction suggests a unique binding interface and translocation mechanism.This work was done in collaboration with the team of Laurent Terradot (IBCP) who coordinated the project.

Structural insights into Helicobacter pylori CagA interaction with β1 integrin. Kaplan Türköz B, Jiménez-Soto LF, Dian C, Ertl C, Remaut H, Louche A, Tosi T, Haas R and Terradot L. Proc. Natl. Acad. Sci. ;109(36):14640-5.

A new method for understanding conformational exchange processes in solid protein samples: application to hidden states in a cristalline protein

Numerous biological processes, such as catalysis or allostery, critically depend on the dynamic process between various protein states. Often, the actual functional state is not the predominant state, but different conformations, in exchange with the lowest-energy state.
While the predominant states can be characterized by classical structural biology (e.g. X-ray diffraction), investigating such hidden states is difficult.
We have developed a new nuclear magnetic resonance (NMR) method that allows characterizing such states. In contrast to existing methods, our approach is applicable to solid protein samples, such as amyloid fibers. In a first application, we could reveal hidden conformational states in a cristalline protein, and show how the cristalline environment impacts motion.

Site-Resolved Measurement of Microsecond-to-Millisecond Conformational-Exchange Processes in Proteins by Solid-State NMR Spectroscopy. Tollinger M, Sivertsen AC, Meier BH, Ernst M, Schanda P. J Am Chem Soc. 2012 Sep 12;134(36):14800-7

Neutron scattering explains how myoglobin can perform without water

Proteins do not need to be surrounded by water to carry out their vital biological functions, according to scientists from the Institut de Biologie Structurale (IBS) in Grenoble, the University of Bristol, the Australian National University, the Institut Laue Langevin and the Forschungszentrum Jülich, in Germany. In a new paper published in the Journal of the American Chemical Society , the team used a state of the art neutron scattering technique to demonstrate that when myoglobin, an oxygen-binding protein found in the muscle tissue of vertebrates, is enclosed in a sheath of surfactant molecules, it moves in the same way as when it is surrounded by water. These motions are essential if a protein is to perform its biological function, and their findings make proteins a viable material for use in new wound dressings or even as chemical gas sensors.

Press release

A Polymer Surfactant Corona Dynamically Replaces Water in Solvent-Free Protein Liquids and Ensures Macromolecular Flexibility and Activity. François-Xavier Gallat, Alex P. S. Brogan, Yann Fichou, Nina McGrath, Martine Moulin, Joachim Wuttke, Stephen Mann, Giuseppe Zaccai, Colin J. Jackson, Adam W. Perriman, and Martin Weik. Journal of the American Chemical Society;134(32):13168-71.

Contamination from affinity column: a new villain in the world of membrane protein crystallization

Strep-Tactin tetramerCrystallization attempts of a chloroplast ATP/ADP transporter from plants led to the crystallization of a contaminant, Strep-Tactin, a triple streptavidin mutant. Trace amounts of Strep-Tactin are sufficient to crystallize and, despite the very small size of the crystals, the structure of Strep-Tactin in complex with desthiobiotin could be solved to 1.9 Å resolution. Due to a strong interaction between streptavidin and a peptide (Strep-Tag), variants of this protein are routinely used for affinity chromatography. We have demonstrated that several detergents used for membrane protein solubilization, solubilize as well Strep-Tactin monomers from the resin used for purification. Therefore, one has to be very cautious with the use of Strep-Tags for the purification of membrane proteins for crystallization.

Contamination from affinity column: a new villain in the world of membrane protein crystallization. R. Panwar, A. Deniaud and E. Pebay-Peyroula. Acta Crystallogr D Biol Crystallogr. 2012 Oct;68(Pt 10):1272-7

A key cell cycle kinase of the pneumococcus

Eukaryotic-like serine/threonine-kinases (Stk) are involved in the regulation of a variety of physiological processes in bacteria. Deletion of the unique Stk (StkP) in the pneumococcus causes morphological aberrations suggesting a role in the division of this pathogenic bacterium. Construction of pneumococcus mutant strains lacking StkP or expressing various forms of the protein demonstrated that the kinase activity and the extracellular and cytoplasmic domains are required for the division. Over the course of this work, carried out in collaboration with Christopher Grangeasse (IBCP, UMR5086, Lyon) who initiated the project, we obtained pneumococcus bearing original shapes: round, elongated, chain-forming or lacking a functional septa. One of the targets of StkP was identified and, when not phophortylated by StkP, causes the same cellular disturbances. StkP is a multifunctional protein that plays crucial functions in pneumococcus cell shape and division.

Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae. Fleurie A, Cluzel C, Guiral S, Freton C, Galisson F, Zanella-Cleon I, Di Guilmi AM, Grangeasse C. Mol Microbiol.;83(4):746-58. doi: 10.1111/j.1365-2958.2011

Better understanding of the role of the NS5A protein during hepatitis C virus infection

The NS5A protein of hepatitis C virus plays an important role in virus replication and particle formation. In collaboration with the research group of D. Willbold (FZ Jülich, D) we have investigated the structure and dynamics of an intrinsically disordered fragment (188 residues) of NS5A comprising interaction sites with a large number of other viral and host proteins. Our study reveals the presence of transient a-helical structures in 3 regions of the peptide sequence that are partly stabilized by long-range tertiary interactions. Two of these transient a-helices form a non-canonical binding motive which allows low affinity binding to the SH3 domain of the tumor suppressor protein Bin-1. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection.

Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A. Sophie Feuerstein, Zsofia Solyom, Amine Aladag, Adrien Favier, Silke Hoffmann, Dieter Willbold, Bernhard Brutscher. Journal of Molecular Biology ; 420(4-5):310-23

Structure of ExoU, toxin of the human pathogen Pseudomonas aeruginosa

The human pathogen Pseudomonas aeruginosa employs its type III secretion system in order to initiate acute infection. One of the toxins injected into cells by this system, ExoU, causes massive cell damage rapidly through phospholipolysis of target membranes. Here, we have solved the structure of ExoU in complex with a partner molecule and identified that it folds into three independent domains. Fluorescence microscopy experiments indicate that once injected, the cell attempts to clear the toxin from the cytosol by introducing it into the endosomal pathway, but this defense action is not quick enough and membranes get degraded in few minutes by ExoU’s phospholipase action, explaining the aggressiveness of Pseudomonas strains that express this toxin.

Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa. Gendrin C, Contreras-Martel C, Bouillot S, Elsen S, Lemaire D, Skoufias DA, Huber P, Attree I, Dessen A. PLoS Pathogens 8, e1002637.

Dynamics of a bacterial multidrug ABC transporter in the inward and outward facing conformations

The study of membrane proteins remains a challenging task and approaches to unravel their dynamics are scarce. Here, we applied H/D exchange coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-Binding Cassette transporter, BmrA, in the inward (resting state) and outward (ATP-bound) facing conformations. Trypsin digestion and global or local H/D exchange support the transition between inward and outward facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster H/D exchange than in the closed state. This shows that these two sub-domains are very flexible in this conformation. Also, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of H/D exchange coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.

Dynamics of a bacterial multidrug ABC transporter in the inward and outward facing conformations. Mehmood, S., Domene, C., Forest, E. & Jault, J.-M. Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10832-6

(IscS-IscU)2 Complex Structures Provide Insights into Fe2S2 Biogenesis and Transfer

The center of attention: IscS cysteine desulfurases and IscU scaffolds are involved in biological iron-sulfur cluster assembly. The X-ray structure of an anaerobically produced, mutated (Fe(2) S(2) -(IscS-IscU(D35A) ))(2) complex reveals a cluster coordinated by three IscU cysteines and the IscS active cysteine. In air-exposed crystals the cluster is oxidized to an Fe(2) S-S center; D35 is essential for complex dissociation.

(IscS-IscU)2 Complex Structures Provide Insights into Fe2S2 Biogenesis and Transfer. Elodie N. Marinoni, Jaim S. de Oliveira, Yvain Nicolet, Estella C. Raulfs, Patricia Amara, Dennis R. Dean, and Juan C. Fontecilla-Camps. Angewandte Chemie International Edition;51(22):5439-42

7th IBS scientific day and 20th anniversary

On June 08, 2012, IBS organized its 7th Science Day, which brought together most of the staff (about 200 people) in the Weil Amphitheater on the campus. Former permanent people were also invited as IBS celebrates its 20th anniversary in 2012.
Eight lectures were given by young researchers and PhD students participated through flashs & poster presentations. The poster price was awarded to Philippe Jules (Pneumococcus group).
A conference by Juan Fontecilla and Joseph Zaccai was the occasion to explain the reasons that led to the creation of the IBS and celebrate people who contributed to its reputation.
On the occasion of its 20th anniversary, a festive evening was also organized in the nearby EVE room.

Conférences Séances posters
Pause déjeuner à EVE Eva Pebay-Peyroula, directrice de l'IBS avec Michel Van der Rest ancien directeur et Richard Wade ancien chef de labo
L'imagination des doctorants à l'épreuve même pendant la soirée !

Note that the IBS will held a ‘Midi Minatec’ conference on October 05, 2012 to celebrate 20 Years of research with all its partners.

Detailed Characterisation of the Conformational Energy Landscape of the SH3C domain of CD2AP by NMR

A complete description of the relationship between structure and function of proteins requires an understanding of the nature and role of conformational dynamics. The nature and amplitude of conformational fluctuations on physiological important time scales, from nanosecond to millisecond, remain an open and important question. In order to obtain insight into this problem, the Protein Dynamics and Flexibility group has developed two orthogonal approaches based on the measurement of residual dipolar couplings (RDCs) by NMR. One involves the analytical modeling of the dynamics of each amino acid where the other relies on accelerated molecular dynamics simulations.
These two methods have been applied to study the dynamics of the SH3C domain of the CD2 Associated Protein (CD2AP). More than 1900 experimental RDCs were measured and combined with spin relaxation rates to arrive at a comprehensive and convergent description of the conformational dynamics of this protein at atomic resolution on time scales ranging from pico to milliseconds. This precise description of the conformational energy landscape of the protein highlights fluctuations in the interaction sites of this protein with correlation times from nano- to microseconds – fluctuations which so far have been invisible to other approaches. Studies of the conformational behaviour of this protein in complex with its interaction partner Ubiquitin will allow for a better understanding of the physiological role of this dynamics.

Multi-Timescale Conformational Dynamics of the SH3 Domain of CD2-Associated Protein using NMR Spectroscopy and Accelerated Molecular Dynamics. Salmon L, Pierce L, Grimm A, Ortega Roldan JL, Mollica L, Jensen MR, van Nuland N, Markwick PR, McCammon JA, Blackledge M. Angew Chem Int Ed Engl.; 51(25):6279.

Putting a stop to L,D-transpeptidases

β-lactams inhibit the biosynthesis of the principal component of the bacterial cell wall, the peptidoglycan, through the inhibition of the D,D-transpeptidase enzymes (also called PBPs) that are responsible for its assembly. In certain cases as for dormant forms of tuberculosis bacilli, L,D-transpeptidases (Ldts), a non-related class of enzyme, can bypass this deficiency and allow the bacterial survival under antibiotic pressure. Then, only the carbapenem antibiotic family keeps a bactericidal activity. To further the understanding of the specificity of Ldts towards carbapenems the Biomolecular NMR Spectroscopy group at IBS, in collaboration with researchers at the Centre de Recherche des Cordeliers in Paris, studied the structure and dynamics of various forms of the Bacillus subtilis Ldt enzyme. They show that the active site cysteine is involved in a catalytic triad in the free enzyme, that the non-covalent binding of β-lactams is weak and is not specific to carbapenems, and that the covalent modification of the catalytic cysteine induces ms-time scale flexibility at the active site. Altogether these results suggest that the nucleophilic attack of the β-lactam carbonyl by the cysteine is the key step in the enzymatic specificity.

Dynamics induced by β-lactam antibiotics in the active site of Bacillus subtilis L,D-transpeptidase. Lecoq L, Bougault, C., Hugonnet J.-E., Veckerlé C., Pessey O., Arthur, M., Simorre J.-P. Structure; 20(5):850-61.

Real-Time NMR Characterization of Structure and Dynamics in a Transiently-Populated Protein Folding Intermediate

Structural biology generally focuses on studies of the highly populated ground states (native states) of proteins. However, it has long been recognized that higher energy structures can play important functional roles, or may be responsible for protein misfolding and fibril formation leading to various diseases. In our study, we demonstrate the capability of real-time high-field multidimensional NMR spectroscopy to provide site-resolved information on the structure and dynamics of protein states that are separated from the ground state by energy barriers of > 10 kcal/mol. We demonstrate this approach for a folding intermediate of the amyloidogenic human protein b2-microglobuline that that is supposed to be responsible for fibril formation in the joints and connective tissues of patient undergoing long-term dialysis.

Real-Time NMR Characterization of Structure and Dynamics in a Transiently-Populated Protein Folding Intermediate. Enrico Rennella, Thomas Cutuil, Paul Schanda, Isabel Ayala, Vincent Forge, and Bernhard Brutscher. Journal of the American Chemical Society, 2012 May 16;134(19):8066-9. Epub 2012 May 7.

A combination of methods to develop a high resolution description of the unfolded state of the protein ubiquitin

Nuclear magnetic resonance (NMR) spectroscopy is increasingly recognised as the most powerful experimental tool for studying protein folding and stability. In a recent study researchers at the IBS used extensive experimental NMR data, in combination with small angle scattering and a unique tool for the description of dynamic conformational equilibria, to develop a high resolution description of the unfolded state of the protein ubiquitin, providing sequence specific details of the interaction between the denaturant and the protein. In related studies the same methodology was applied, in collaboration with researchers at the Goethe university in Frankfurt, to understand the folding pathway of the protein Lysozyme.

Sequence-specific mapping of the interaction between urea and unfolded ubiquitin from ensemble analysis of NMR and small angle scattering data. Huang JR, Gabel F, Jensen MR, Grzesiek S, Blackledge M. J Am Chem Soc. 2012 Mar 7;134(9):4429-36.

Modulation of structure and dynamics by disulfide bond formation in unfolded States. Silvers R, Sziegat F, Tachibana H, Segawa S, Whittaker S, Günther UL, Gabel F, Huang JR, Blackledge M, Wirmer-Bartoschek J, Schwalbe H. J Am Chem Soc. 2012 Apr 18;134(15):6846-54.

Rational design of a remarkably efficient cyan fluorescent protein

Researchers from the IBS (Institut de biologie structurale, CEA-CNRS-UJF) and the ESRF (European Synchrotron Radiation Facility), in collaboration with groups from the Universities of Amsterdam and Oxford, have managed to design a new cyan fluorescent protein (CFP) whose fluorescence efficiency is unmatched among this family of proteins, using a combination of structural biology and genetic engineering.
Thanks to their natural fluorescence, fluorescent proteins can be readily located and tracked in organisms. They constitute very good biochemical and biological markers and are essential for certain techniques of cell imaging. Cyan and yellow fluorescent proteins (CFP and YFP) are used in the so-called FRET technique to probe protein-protein interactions and protein conformational changes in cell signalling cascades. Unfortunately, CFPs have long suffered from a weak level of fluorescence, which contributes to obscure experimental results. Researchers from Grenoble, Oxford and Amsterdam have teamed up to understand and improve these proteins. Using the synchrotron radiation of the ESRF, the Grenoble team has uncovered subtle details of their structural dynamics, which were used to determine which part of the protein had to be modified to increase the level of fluorescence. The team in Amsterdam then used their innovative high-throughput screening technique based on fluorescence lifetime microscopy to identify improved proteins. In the end, the scientists managed to design and characterize a new CFP called mTurquoise2 whose fluorescence efficiency is unmatched for such proteins. mTurquoise2 will now allow researchers to study protein-protein interactions in live cells with an unprecedented level of sensitivity and to perform experiments whose results would have been hidden in noise before.

Communiqué de presse/Press release

Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%, J. Goedhart, D. von Stetten, M. Noirclerc-Savoye, M. Lelimousin, L. Joosen, M. A. Hink, L. van Weeren, T. W. Gadella Jr, and A. Royant, Nat. Commun. 2012 Mar 20;3:751

The IBS actor of INSTRUCT, the European integrated biology infrastructure

Breakthroughs in biomedical science are a step closer, with the launch, on February 23rd, 2012, of a new distributed research infrastructure for the science of structural biology. The launch of ‘Instruct’ will give academic and commercial scientists across Europe access to a full portfolio of integrated technologies, thanks to the collaboration of leading european structural biology research institutes.

Instruct provides access to some of the most advanced technology in the world. A full catalogue of the accessible technology is available on the Instruct Hub at The Hub includes adverts for jobs and academic programmes, a comprehensive calendar of events and discussion forums, with plans for virtual workspaces for collaborative projects in the near future. The Hub is also home to a number of networks of scientists with a common interest or objective.

Grenoble (PSB) and Strasbourg (IGBMC) are the two French Instruct centers among 15 Instruct centers. The Institut de Biologie Structurale provides access to high-tech instruments and cutting-edge know-how through a number of platforms (Cryo EM, MP3 et NMR).

A peptide to mimic a sugar and inhibit the entry of HIV into cells

The entry of HIV into host cells is based on the interaction between the viral protein gp120 with the CD4 receptor and GPCRs, CCR5 and/or CXCR4. This dual interaction represents a pharmacological target of interest for strategies aimed at inhibiting viral entry.
In this perspective, the SAGAG group at the IBS solubilized CCR5 and CXCR4 in a cocktail of lipids and detergents, to maintain their functional recognition, and measured the interactions between these receptors and gp120 by SPR.
Moreover, our previous work has shown that heparan sulfate (HS) binds to gp120 and interferes with viral entry. The complexity of the HS does not allow easy identification of molecular determinants involved in this interaction, therefore, peptides containing sulfotyrosines (whose number and position can be easily modified) were generated to mimic these polysaccharides.
By combining these two approaches (measurement of gp120/GPCR interactions and generation of oligosaccharide mimetics), a new molecule (mCD4-P3YSO3) was developed in collaboration with the Pasteur Institute and the University of Orsay. This molecule inhibits the interaction of gp120 with CD4, CCR5 and CXCR4 and blocks the replication of clinical HIV-1 isolates with an IC50 as low as 0.5 nM. These results thus favor the development of this molecule as a novel entry inhibitor of HIV-1.

A synthetic heparan sulfate-mimetic peptide conjugated to a mini CD4 displays veryhigh anti-HIV-1 activity independently of coreceptor usage. Connell B.J., Baleux F., Coic YM., Clayette P., Bonnaffé D and Lortat-Jacob H. Chemistry & Biology 19 (1) 131-139 (2012)

Molecular mechanisms of the biogenesis of Streptococcus pneumoniae pilus

Model of the assembly of S. pneumoniae pilus.
RrgB forms the pilus polymer fiber. The adhesin RrgA, promoting pilus interaction with the host receptors, is associated with the end of the pilus but also along the fiber. Multimerization of RrgA should also increase the avidity of recognition. RrgC is likely to anchor the pilus in the bacterial cell wall. The function and the proposed sequence of the sortase activity during the pilus construction is described (El Mortaji, Biochemistry, 2012).

Streptococcus pneumoniae is a major human pathogen, responsible for ear infections, pneumonia, septicemia and meningitis. Recently pilus-like structures were identified on the surface of S. pneumoniae, which play an important role in the initial stages of colonization of host tissues. Six genes are involved in the formation of this structure. Three of them encode structural proteins or pilins (RrgA, RrgB and RrgC) whereas three other genes code for enzymes called sortases, catalyzing the covalent association of pilins (SrtC-1, SrtC-2 and SrtC-3 ). The structural characterization of pilins RrgA, RrgB and RrgC was completed owing a collaboration between the pneumococcus and the Bacterial Pathogenesis Groups of the IBS. It revealed the presence of a new type of posttranslational modification: intramolecular bridges between Lys-Asn residues. The stabilizing effect on each pilins of these unusual covalent bonds has been established. Furthermore, the crystallographic structures of RrgA and RrgB has elucidated some aspects of the pilus assembly process and its adhesive properties. To define the substrate specificity of each of the three sortases, we have developed a platform of co-expression of pilins and sortases in the E. coli recombinant system. This study identified the specific sortases catalysing the formation of complexes between pilins and sortases. Overall this work has revealed key insights in our understanding of the biogenesis of the pilus in S. pneumoniae allowing us to propose a new model of pilus assembly.

- Manzano C., Contreras-Martel C., El-Mortaji L., Izore T., Fenel D., Vernet T., Schoehn G., Di Guilmi A.M., Dessen A. (2008). Structure, 16, 1-11.
- Manzano C., Izore T., Job V., Di Guilmi A.M., Dessen A. (2009) Biochemistry, 48, 10549-10557.
- El Mortaji L., Terrasse R., Dessen A., Vernet T., Di Guilmi A.M. (2010) J. Biol. Chem. 285, 12405-12415.
- Izoré T., Contreras-Martel C., El-Mortaji L., Manzano C., Terrasse R., Verne.t T., Di Guilmi A.M., Dessen A. (2010) Structure 18, 106-115.
- El Mortaji L., Fenel D., Vernet T., Di Guilmi A.M. (2012) Biochemistry. Under press
- El Mortaji L., Contreras-Martel C., Moschioni M., Ferlenghi I., Manzano C., Vernet T., Dessen A., Di Guilmi A.M. (2012) Biochemical J. 441, 833-841

Inhibition of HIV infection in human uterine tissue samples

More than 18 months ago, our team has contributed, in the context of a European network, to the demonstration that some molecules inhibiting DC-SIGN, that are produced within our network (glycomimic dendrons), had the potency to block DC-SIGN-mediated HIV trans-infection of T lymphocytes. DC-SIGN is a receptor of dendritic cells present in epithelium and mucosa that is hijacked by HIV in order to be transmitted through the genital mucosal barrier.
The work that we publish these days in AIDS represents a step forward in this investigation. Indeed, beyond the trans-infection, this is now a direct inhibition of HIV infection that our compounds enable, not only on cellular preparations but also now on human genital tissue samples (cervical explants). Besides, we also revealed that our inhibitors, through their interaction with DC-SIGN, lead to an increase of the chemokines MIP1-α, MIP1-β and RANTES and therefore trigger an intracellular signaling. Interestingly, those chemokines are the natural ligands of CCR5, the co-receptor of HIV on its target cells. Therefore a boost of their production and release could, by blocking their cellular receptor, contribute to the observed infection inhibition.
This study validates our strategy for new molecules that could be used as microbicides. We demonstrate an inhibition specific to DC-SIGN, the receptor “hijacked” by HIV, without affecting langerin, an analogous receptor described to contribute to HIV elimination.
Our consortium keeps on trying to improve those inhibitors. A new generation of molecules with a better affinity is already emerging from our laboratories.

A glycomimetic compound inhibits DC-SIGN-mediated HIV infection in cellular and cervical explant models. Berzi A, Reina JJ, Ottria R, Sutkeviciute I, Antonazzo P, Sanchez-Navarro M, Chabrol E, Biasin M, Trabattoni D, Cetin I, Rojo J, Fieschi F, Bernardi A, Clerici M. AIDS. 2012 Jan 14;26(2):127-137.