Structure and Dynamics of Nucleoproteic and Membrane Assemblies


Group Leaders: Christian Roumestand and André Padilla

Team 1 comprises experienced researchers in structural biology (Biochemistry, NMR, Crystallography and SAXS) in order to address challenging biological questions. The aim of the team is to take advantage of this broad spectrum of complementary skills to study complex biological systems from an interdisciplinary perspective. The capacity to master several structural biology techniques will ensure the proper development of the scientific challenges. The activities of the team in the last years can be divided in two axes : structural biology of infectious diseases, and High-Pressure NMR and protein unfolding. Important achievements have been accomplished and these broad research lines will be continued in the following years in a more interdisciplinary and integrative approaches will be used to reach better structural and dynamic description of the biomolecular systems targeted.

NMR Platform


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Coordinators: André Padilla & Christian Roumestand

Technical manager: Karine de Guillen


Researchers and Engineers of the NMR team are involved in the platform projects.

 NMR people : De Guillen Karine, Déméné Hélène, Barthe Philipe, Yang Yinshan, Cohen-Gonsaud Martin



NMR structure of RBPMS2 N-ter

NMR Platform Staff : Yang Y.       Pipeline from the CBS BioInformatics Platform

Protein Size : 2*92 a.a. 15N and 13C labeled

In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. For the first time, the NMR expriments show that the key protein RBPMS2 homodimerizes through a particular sequence motif located in its RRM domain.

rbpm1 rbpm2

External Contact : P. de Santa Barbara, This email address is being protected from spambots. You need JavaScript enabled to view it. INSERM U1046, Université Montpellier

Sagnol S, Yang Y, Bessin Y, Allemand F, Hapkova I, Notarnicola C, Guichou JF, Faure S, Labesse G, de Santa Barbara P (2014) Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity, Nucleic Acids Res () . PMID = 25064856


TGAM_1934, an ORFan protein from the archeon Thermococcus gammatolerans.

NMR Platform Staff: de Guillen K., Yang Y. & Roumestand C.

Protein Size : 148 a.a. 15N and 13C labeled

ORFans are proteins found when annotating new genomes by comparative genomics. TGAM_1934, a 122 amino acid polypeptide from the hyper-radioresistant T. gammatolerans archaeon, was selected as the most abundant hypothetical protein amongst the low molecular weight proteome. The NMR results highlight the potential of structural proteogenomics, i.e. prioritizing ORFan targets for structure determination based on quantitative proteomics data.

tgamhsqc tgam 


External Contact : Jean Armengaud , This email address is being protected from spambots. You need JavaScript enabled to view it. CEA, DSV, IBEB, Bagnols-sur-Cèze

Yang YS, Fernandez B, Lagorce A, Aloin V, De Guillen KM, Boyer JB, Dedieu A, Confalonieri F, Armengaud J, Roumestand C (2014) Prioritizing targets for structural biology through the lens of proteomics: The archaeal protein TGAM_1934 from Thermococcus gammatolerans, Proteomics. PMID = 25359407


NMR Structure of an Antimicrobial peptide

NMR Platform Staff : André Padilla                   pipeline from CBS RX platform


Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics. Aedesin has a helix-bend-helix structure typical for a member of the family of alpha-helix anti-microbial peptides.


Spectra overlay : TOCSY (green) NOESY (yellow)


Surface coloured by the charge, blue for positive red for negative and grey for hydrophobic residues


External Contact: D. Missé (MIVEGEC, UMR224 IRD/CNRS/UM) Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Godreuil S, Leban N, Padilla A, Hamel R, Luplertlop N, Chauffour A, et al. Aedesin: Structure and Antimicrobial Activity against Multidrug Resistant Bacterial Strains. PLoS ONE 2014;9:e105441. doi:10.1371/journal.pone.0105441



 Fragment based drug discovery – NMR screening by STD

NMR Platform Staff : André Padilla                                     Pipeline to CBS RX platform

Protein Size: 22 kDa, Unlabeled @ 30 µM

By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno. By use of NMR, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated.





STD spectra


XRay Structure of the ARNO-Sec7-FC7 complex

External Contact: J. Rouhana (IBMM Montpellier)

Rouhana J, Hoh F, Estaran S, Henriquet C, Boublik Y, Kerkour A, et al. Fragment-Based Identification of a Locus in the Sec7 Domain of Arno for the Design of Protein-Protein Interaction Inhibitors. J Med Chem 2013. doi:10.1021/jm4009357.


NMR Structure of an highly specific tumor-targeting miniprotein

NMR Platform Staff : Philippe Barthe

In this study, the scaffold properties of the two-disulfide-stabilized miniprotein Min-23 (DLL-Rib) were exploited to generate new affinity reagents against the angiogenesis marker Delta-like Ligand 4 (DLL4) performing ribosome display. In both in vitro and in vivo studies it was shown, that only the properly folded miniprotein specifically bond to the target DLL4. These characteristics allowed for imaging of tumor with positron emission tomography applying radiolabeled miniproteins.

min-23 3

Zoom region of the DLL-Rib peptide {1H-13C} HSQC. Cystein's beta-carbons are highlighted in green.

min-23 4

Ribbon 3D structure of the DLL-Rib peptide. Disulfide bridges are coloured in orange.

External Contact: U. Haberkorn (University of Heidelberg) This email address is being protected from spambots. You need JavaScript enabled to view it.

Zoller F, Markert A, Barthe P, Hebling U, Altmann A, Lindner T, Mier W, Haberkorn U. A disulfide-constrained miniprotein with striking tumor-binding specificity developed by ribosome display. Angew Chem Int Ed Engl. 2013 Nov 4;52(45):11760-4. doi: 10.1002/anie.201304603.


NMR structure of Bc28.1

NMR Platform Staff: Yang Y. & Roumestand C.

Protein Size : 223 a.a. 15N and 13C labeled

Babesia canis is the agent of the canine babesiosis in Europe. The major merozoite surface antigens of Babesia canis have been described as a 28-kDa membrane protein family, anchored at the surface of the merozoite. The resolution of the structure of Bc28.1 (223 a.a) represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.




Left Panel : 15N - HSQC and zooms of lightly crowded areas


 External Contact : Stéphane Delbecq EA4558, Vaccination Antiparasitaire, Montpellier.


Yang YS, Murciano B, Moubri K, Cibrelus P, Schetters T, Gorenflot A, Delbecq S, Roumestand C (2012) Structural and functional characterization of Bc28.1, major erythrocyte-binding protein from Babesia canis merozoite surface, J Biol Chem 287 (12) 9495-9508


NMR structure of the virulence factor MgtC domain

NMR Platform Staff : Yang Y. & Cohen-Gonsaud M.

Protein Size : 94 a.a. 15N and 13C labeled

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens. To get insights into MgtC functional and structural organization, the structure of the C-terminal domain of M. tuberculosis MgtC was determined by NMR.


mgtc700hs mgtc 


External Contact : Anne-Béatrice Blanc-Potard (CNRS, UMR 5235, Montpellier, France)

Yang Y, Labesse G, Carrere-Kremer S, Esteves K, Kremer L, Cohen-Gonsaud M, Blanc-Potard AB (2012) The C-terminal domain of the virulence factor MgtC is a divergent ACT domain., J. Bacteriol. 194 (22) 6255-63 . PMID = 22984256


NMR Structure of a tumor-targeting chimeric protein

NMR Platform Staff : Philippe Barthe.

We used the disulfide-stabilized miniprotein Min-23 as a molecular scaffold in a phage display approach to generate a combinatorial library for the identification of new affinity functions against the angiogenesis marker delta-like ligand 4 (Dll4). The grafting of the binding domain from miniprotein Min-23 into the sunflower trypsin inhibitor (SFTI-I) peptide scaffold preserved its in vitro and in vivo binding specificity and proteolytic stability. Both the Dll4-binding specificity and the tumor-targeting capability of the rationally designed miniprotein (SFMIN3) were confirmed in vitro and in vivo.


min-23 1

Fingerprint TOCSY of the major (black) and

minor (blue*) forms of SFMIN3 peptide.

min-23 2

Ribbon 3D structure of the SFMIN3 peptide. beta-sheet is represented by purple arrows, disulfide bridge is coloured in orange.

External Contact: U. Haberkorn (University of Heidelberg) Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Zoller F, Markert A, Barthe P, Zhao W, Weichert W, Askoxylakis V, Altmann A, Mier W, Haberkorn U. Combination of phage display and molecular grafting generates highly specific tumor-targeting miniproteins. Angew Chem Int Ed Engl. 2012 Dec 21;51(52):13136-9. doi: 10.1002/anie.201203857.



Structure of the Mycobacterium tuberculosis OmpATb

NMR Platform Staff: Yang Y. & Roumestand C.

Protein Size : 256 a.a. 15N and 13C labeled

The pore-forming outer membrane protein OmpATb from Mycobacterium tuberculosis is a virulence factor shown to be required for acid resistance in host phagosomes. NMR studies show that OmpATb is composed of two independent domains separated by a proline-rich hinge region. The results revealed the N-terminal domain of OmpATb can assemble into multiple oligomeric forms and is responsable to form channels in planar lipid bilayers.


15N-HSQC spectrum 256 a.a.


N-terminal domain structure


HADDOCK model of the heptamer

External Contact : Nathalie Saint (INSERM U1046) Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Yang Y, Auguin D, Delbecq S, Dumas E, Molle G, Molle V, Roumestand C, Saint N (2011) Structure of the Mycobacterium tuberculosis OmpATb protein: a model of an oligomeric channel in the mycobacterial cell wall, Proteins 79 (2) 645-661 . doi: 10.1002/prot.22912.



NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.

NMR Platform staff : Yang Y. & Padilla A.

Protein Size : 98 a.a. unlabeled


The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. On the basis of the 3D structure and structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs.



External Contact : Evelyne BACHERE (IFREMER-CNRS-Université Montpellier, FRE 2626)

Yang Y, Boze H, Chemardin P, Padilla A, Moulin G, Tassanakajon A, Pugniere M, Roquet F, Destoumieux-Garzon D, Gueguen Y, Bachere E, Aumelas A (2009) NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site, Biopolymers 91 (3) 207-220 . PMID = 19107926


NMR structure of the mammalian deoxynucleotide N-hydrolase Rcl protein

NMR Platform Staff : Yang Y. & Padilla A.                      Pipeline to the CBS RX Platform

Protein Size : 2*150 a.a. 15N and 13C labeled

The gene Rcl encodes a deoxynucleoside 5′-monophosphate N-glycosidase that catalyzes the hydrolysis of the N-glycosidic bond of the nucleotide to give deoxyribose 5-phosphate and a nucleobase. The NMR study reveal an important ligand-induced stabilization of the dimer structure of the enzyme which can be used as a new and attractive therapeutic target.


External Contact : P. Alexandre Kaminski (URA CNRS 2128, Institut Pasteur, 75724 Paris)

Yang Y, Padilla A, Zhang C, Labesse G, Kaminski PA (2009) Structural characterization of the mammalian deoxynucleotide N-hydrolase Rcl and its stabilizing interactions with two inhibitors, J Mol Biol 394 (3) 435-447 . PMID = 19822152


Structure and dynamics of RYMV viral encoded P1 protein

NMR Staff : Déméné H., Yang Y.

Coll. : Florence Vignols and Christophe Brugidou (IRD)

The P1 protein is a crucial protein of the RYMV (Rice Mottle Yellow) virus that infects the most productive rice plants in Africa. Using an integrative approach combining X-Ray and NMR spectroscopy at the CBS, we have characterized the structure and revealed the dynamics of theRYMV P1 protein that are linked to the mode of viral activation.


Structure of AntiTerminators


• Structure and interactions of bacterial antiterminators •

Coordinators: Hélène Déméné

The Bacillus substilis LicT antiterminator protein represents the prototype in gram+ bacteries of widespread regulators of the BglG antitermination familiy. LicT regulates expression of the bglS gene and bglPH operons involved in the transport and metabolim of b glucosides. When activated, these transcriptional regulators bind to a ribonucleotidic antiterminator (RAT) sequence in targets mRNAs, preventing the formation of an antitermination hairpin associated with premature arrest of transcription. All members of the LicT family present a modular structure composed of a RNA binding domain (CAT) and two regulatory homologous domains PRD1 and PRD2. These two domains are the subject of concurrent phosphorylations by enzymes of the PTS (phosphoenolpyruvate PhosphoTransferase System) that control the activation (RNA binding capacity) of LicT. We have previously established the conditions of stability of truncated proteins (CAT-PRD1) and proposed a model of activation for these proteins. We now investigate on the structure of the whole protein, either alone or in interaction with its PTS partners. Our collaborators focus on the structural mechanism at the RNA level.


Figure : models of activated (closed ) and inactive (open) LicT-CAT-PRD1. The degree of opening of the RNA binding domain determines the level of RNA binding activity. LICT


NMR Staff : Yang Yinshan

Coll. Nathalie Declerck (CBS)



Structure of GPCR


• Structure and Activation of G-Protein Coupled Receptors •

Coordinator: Hélène Déméné

NMR staff : Hélène Déméné, Yang Yinshan


This work is issued from a close collaboration between the CBS partners and the IGF partners.

G-Protein Coupled Receptors represent the largest class of membrane surface cell receptors involved in signal transduction in eukaryotes. They sense molecules outside the cell and activate signal transduction pathways inside the cell and, ultimately, cellular responses, by coupling with G-proteins. Our research themes focus on the receptors of the vasopressin (V1R and V2R) and on the µ-opioid receptor (µOR).

In the past, we have deciphered the structural features of isolated intracellular loops of V1aR and V2R. We have shown how they fold by themselves, independently from the rest of the receptors, and are also able to recruit intracellular effectors by themselves. We have proposed a method to produce them under a recombinant form mimicking the natural anchoring in the receptor.

We now investigate on the structure-activity of whole GPCRs, taking advantage of the sensitivity of methyl-based NMR. In particular, we have shown that the ligand- and G-protein binding interfaces are weakly coupled in µOR, extending the concept observed before for the ß2 adrenergic receptor by the Kobilka's team. Based on the NMR spectral parameter changes upon interaction of µOR with various agonists as well as with a G-protein mimetic, we could propose a model of event propagation during activation.



GPCR Figure: agonist binding promotes primary structural changes in the first intracellular loop and the H8 helix, whereas conformational adaption in TM5 and TM6 is limited compared to the full-active state obtained in presence of agonist and G-protein.


 Coll. Sébastien Granier, (IGF); Christiane Mendre, (IGF); Bernard Mouillac, (IGF)


Plant pathogens

• Plant Pathogens and Infectious Diseases •

Coordinator: André Padilla

Agropolis International in Montpellier has a large range of expertise in the area of agriculture, food, environmental biology and biodiversity. The CBS teams have engaged since years collaborations with many Agropolis Teams.

- Recognition of avirulence (AVR) proteins by plant immune Receptors.

Plant resistance to microbial pathogens is a complex process relying on two major levels of resistance triggered by distinct types of plant receptors. Beside the first line of immunity, in which microbial molecules, such as bacterial flagellin or cell wall components of the pathogen are perceived, leading to plant resistance, the second layer of plant immunity relies on the recognition of certain pathogen-derived effectors by so-called plant resistance proteins (R) encoded by R genes. Effectors that are specifically recognized are called Avirulence proteins (Avr) and induce a plant Effector-Triggered Immunity. We use the rice blast model system to investigate R protein function and AVR protein recognition. Rice blast caused by the ascomycete fungus Magnaporthe oryzae is the most important rice disease wide worldwide and as such is a serious economic problem and a major threat for food security and health care linked to pesticides usage. Our collaboration (INRA Montpellier) resulted in the generation of NMR structures for the M. oryzae effectors AVR1-CO39 and AVR-Pia and the project aims at identifying the structure of R rice immune receptors.



The molecular details of R binding are elucidated In vitro and In vivo to validate structural models of Avr recognition and to investigate structure-function relations. Plant diseases are among the most important problems in agriculture and the use of disease resistance (R) genes is a key strategy for sustainable crop protection.



NMR Staff : Karine deGuillen

Coll. Thomas Kroj (INRA)