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Integrative Biological NMR

Alain Milon

Group Leader

Our group is dedicated to NMR development and its applications in structural biology and pharmacology. We are particularly interested in NMR of membranes and membrane proteins (outer membrane proteins and G-proteins coupled receptors), protein – nucleic acid complexes, and NMR based ligand screening as a member of the PICT platform (integrated
ligand screening platform of Toulouse).

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The IPBS NMR group possesses all the expertise and equipment to pursue ambitious projects in NMR structural biology with strong implications in pharmacology. Our strategy is to maintain a high level of expertise by continuously exploring new avenues (such as “Cellular ssNMR spectroscopy”, macromolecular dynamics and its influence on interactions), while applying them on hot topics in structural biology and pharmacology.
Our current research interest goes into three directions which strongly
cross fertilize each other: Cellular ssNMR of Corynebacteriales, dynamics of activation of opioid receptors by their neuropeptides, protein-DNA/RNA interactions.

This research is “integrative” in the sense that we are interested in large supramolecular complexes and/or proteins structure and dynamics in their native environment.

 

Cellular ssNMR of Corynebacteriales
The overall aim of this project is the delivery of an efficient and generally applicable strategy allowing atom-level structural and dynamical investigations of cell wall-associated membrane proteins from Corynebacteriales suborder (M Renault et al., PNAS 2012)


Dynamics of activation of GPCRs by their neuropeptides.

Since the discovery of the 3D structure of many GPCRs, the scientific focus
moved to their dynamics and activation mechanism. We determined
the KOR-bound structure and dynamics of dynorphin, and are
currently exploring other GPCRs and other states along the activation
pathway. (O’Connor et al., PNAS 2015)


Protein-DNA/RNA interactions
Many fundamental biological processes are regulated by specific protein-DNA or protein-RNA interactions which can be regulated by drugs, making these systems both a challenging avenue for biological NMR and a rich source of
pharmacological targets (V Gervais et al., J Biomol NMR 2013).

 

Main Publications

 

• Saurel O et al. (2017) Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution, J Amer Chem Soc 139, 1590-1597.


• Carel C et al. (2017) Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane, Proc Natl Acad Sci USA 114, 4231-4236.


• O’Connor C et al. (2015) NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor, Proc Natl Acad Sci U S A 112, 11852-11857.


• Bartolami E et al. (2015) Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters, Angew Chem Int Ed Engl 54, 10183-10187.


• Campagne S et al. (2012) Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1, Nucleic Acids Research 40, 9927-9940.

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Outer membrane protein A from K. pneumoniae.

We have determined its 3D structure and dynamics by NMR in detergent micelles (M Renault et al., J Mol Biol 2009) and in liposomes (I Iordanov et al., BBA 2012). Our current objective is to characterize outer membrane proteins in their native environment by cellular solid state NMR. This includes a number of porins from Corynebacterium and Mycobacterium cell walls. Solid state NMR also allows describing the outer membrane lipids supramolecular organization and dynamics.

 

Scientists

Alain Milon

Professor - University of Toulouse

Georges Czaplicki

Professor - University of Toulouse

Matthieu Chavent

CNRS Research associate

Pascal Demange

CNRS Research director

Virginie Gervais

CNRS Research associate

Isabelle Muller

Associate Professor - University of Toulouse

Valerie Réat

CNRS Research associate

Justin Teissié

Emeritus CNRS research director

Research Assistants

Nathalie Doncescu

CNRS Engineer

Pascal Ramos

University of Toulouse Engineer

Olivier Saurel

CNRS Engineer

PhD students

Guillaume Ferre

2018

  • Drozdz, W., Walczak, A., Bessin, Y., Gervais, V., Cao, X. Y., Lehn, J. M., Ulrich, S., and Stefankiewicz, A. R. (2018) Multivalent metallosupramolecular assemblies as effective DNA binding agents, Chemistry.
  • Drozdz, W., Bessin, Y., Gervais, V., Cao, X. Y., Lehn, J. M., Stefankiewicz, A. R., and Ulrich, S. (2018) Switching Multivalent DNA Complexation using Metal-Controlled Cationic Supramolecular Self-Assemblies, Chemistry 24, 1518-1521.

2017

  • Duncan, A. L., Reddy, T., Koldso, H., Helie, J., Fowler, P. W., Chavent, M., and Sansom, M. S. P. (2017) Protein crowding and lipid complexity influence the nanoscale dynamic organization of ion channels in cell membranes, Sci Rep-Uk 7.
  • Cukier, C. D., Tourdes, A., El-Mazouni, D., Guillet, V., Nomme, J., Mourey, L., Milon, A., Merdes, A., and Gervais, V. (2017) NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex, Protein Sci 26, 2240-2248.
  • Carel, C., Marcoux, J., Reat, V., Parra, J., Latge, G., Laval, F., Demange, P., Burlet-Schiltz, O., Milon, A., Daffe, M., Tropis, M. G., and Renault, M. A. M. (2017) Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane, Proc. Natl. Acad. Sci. USA 114, 4231-4236.
  • Saurel, O., Iordanov, I., Nars, G., Demange, P., Le Marchand, T., Andreas, L. B., Pintacuda, G., and Milon, A., Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution, J.A.C.S. 139, 1590-1597 (2017).

2016

  • Menchon, G., Bombarde, O., Trivedi, M., Negrel, A., Inard, C., Giudetti, B., Baltas, M., Milon, A., Modesti, M., Czaplicki, G., and Calsou, P. (2016) Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface, Sci. Rep. 6, 22878 (2016).
  • Hemmann, J. L., Saurel, O., Ochsner, A. M., Stodden, B. K., Kiefer, P., Milon, A., and Vorholt, J. A., The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of alpha- and gamma-Linked Glutamic Acid Residues, J. Biol. Chem. 291, 9042-9051 (2016).
  • Cukier, C. D., Maveyraud, L., Saurel, O., Guillet, V., Milon, A., and Gervais, V., The C-terminal region of the transcriptional regulator THAP11 forms a parallel coiled-coil domain involved in protein dimerization, J. Struct. Biol. 194, 337-346 (2016).
  • Brison, Y., Malbert, Y., Czaplicki, G., Mourey, L., Remaud-Simeon, M., and Tranier, S., Structural Insights into the Carbohydrate Binding Ability of an alpha-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70, ‎J. Biol. Chem. 291, 7527-7540 (2016).

2015

  • Sinnige, T., K. Houben, I. Pritisanac, M. Renault, R. Boelens, and M. Baldus. Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach. J. biomol. NMR 61, 321-332 (2015)
  • O’Connor, C., K. L. White, N. Doncescu, T. Didenko, B. L. Roth, G. Czaplicki, R. C. Stevens, K. Wuthrich, and A. Milon. NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor. PNAS 112, 11852-11857 (2015).
  • Berthoumieu, O., P. H. Nguyen, M. P. Castillo-Frias, S. Ferre, B. Tarus, J. Nasica-Labouze, S. Noel, O. Saurel, C. Rampon, A. J. Doig, P. Derreumaux, and P. Faller. Combined experimental and simulation studies suggest a revised mode of action of the anti-Alzheimer disease drug NQ-Trp. Chemistry 21, 12657-12666 (2015).
  • Bartolami, E., Y. Bessin, V. Gervais, P. Dumy, and S. Ulrich. Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters. Angew. Chemie 54, 10183-10187 (2015).

2014

  • Sinnige, T., M. Weingarth, M. Renault, L. Baker, J. Tommassen, and M. Baldus. Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility. J. Mol. Biol. 426, 2009-2021 (2014).
  • Planchard, N., E. Point, T. Dahmane, F. Giusti, M. Renault, C. Le Bon, G. Durand, A. Milon, E. Guittet, M. Zoonens, J. L. Popot, and L. J. Catoire. The use of amphipols for solution NMR studies of membrane proteins: advantages and constraints as compared to other solubilizing media. J. Membr. Biol. 247, 827-842 (2014).
  • Nars, G., O. Saurel, F. Bordes, I. Saves, M. Remaud-Simeon, I. Andre, A. Milon, and A. Marty. Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems. Protein Expr. Purif. 101, 14-20 (2014).
  • Nakatani, Y., N. Ribeiro, S. Streiff, M. Gotoh, G. Pozzi, L. Desaubry, and A. Milon. Search for the Most ’primitive’ Membranes and Their Reinforcers: A Review of the Polyprenyl Phosphates Theory. Orig Life Evol Biosph 44, 197-208 (2014).
  • Gater, D. L., O. Saurel, I. Iordanov, W. Liu, V. Cherezov, and A. Milon. Two classes of cholesterol binding sites for the beta2AR revealed by thermostability and NMR. Biophys. J. 107, 2305-2312 (2014).
  • Bissaro, B., O. Saurel, F. Arab-Jaziri, L. Saulnier, A. Milon, M. Tenkanen, P. Monsan, M. J. O’Donohue, and R. Faure. Mutation of a pH-modulating residue in a GH51 alpha-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products. Biochim Biophys Acta. 1840, 626-636 (2014).
  • Abot, A., C. Fontaine, M. Buscato, R. Solinhac, G. Flouriot, A. Fabre, A. Drougard, S. Rajan, M. Laine, A. Milon, I. Muller, D. Henrion, M. Adlanmerini, M. C. Valera, A. Gompel, C. Gerard, C. Pequeux, M. Mestdagt, I. Raymond-Letron, C. Knauf, F. Ferriere, P. Valet, P. Gourdy, B. S. Katzenellenbogen, J. A. Katzenellenbogen, F. Lenfant, G. L. Greene, J. M. Foidart, and J. F. Arnal. The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor alpha modulation, uncoupling nuclear and membrane activation. EMBO Mol Med 6, 1328-1346 (2014).

2013

  • Renault, M., L. Shintu, M. Piotto, and S. Caldarelli. Slow-spinning low-sideband HR-MAS NMR spectroscopy: delicate analysis of biological samples. Scientific reports 3, 3349 (2013).
  • Renault, M., J. Garcia, T. N. Cordeiro, M. Baldus, and M. Pons. Protein oligomers studied by solid-state NMR—the case of the full-length nucleoid-associated protein histone-like nucleoid structuring protein. FEBS J. 280, 2916-2928 (2013).
  • Gater, D. L., V. Reat, G. Czaplicki, O. Saurel, A. Milon, F. Jolibois, and V. Cherezov. Hydrogen bonding of cholesterol in the lipidic cubic phase. Langmuir : the ACS journal of surfaces and colloids 29, 8031-8038 (2013).
  • Rath, P., Saurel, O., Czaplicki, G., Tropis, M., Daffe, M., Ghazi, A., Demange, P. et Milon, A. Cord factor (trehalose 6,6’-dimycolate) forms fully stable and non-permeable lipid bilayers required for a functional outer membrane, BBA - Biomembranes 1828, 2173-2181 (2013).
  • Gervais, V., Campagne, S., Durand, J., Muller, I. et Milon, A. NMR studies of a new family of DNA binding proteins: the THAP proteins, J. Biomol. NMR 56, 3-15 (2013).
  • Cala, O., Remy, M. H., Guillet, V., Merdes, A., Mourey, L., Milon, A., and Czaplicki, G. Virtual and biophysical screening targeting the gamma-tubulin complex—a new target for the inhibition of microtubule nucleation, PLoS One 8, e63908 (2013).
  • Arab-Jaziri, F., Bissaro, B., Dion, M., Saurel, O., Harrison, D., Ferreira, F., Milon, A., Tellier, C., Faure, R., and O’Donohue, M. J. Engineering transglycosidase activity into a GH51 alpha-l-arabinofuranosidase, N. Biotechnol. 30, 536-544 (2013).
  • Gater, D. L., Reat, V., Czaplicki, G., Saurel, O., Jolibois, F., Cherezov, V. et Milon, A. Hydrogen bonding of cholesterol in the lipidic cubic phase. Langmuir 29, 8031-8038 (2013).
  • Rath, P., Saurel, O., Tropis, M., Daffe, M., Demange, P., and Milon, A. NMR localization of the O-mycoloylation on PorH, a channel forming peptide from Corynebacterium glutamicum, FEBS Lett. 587, 3687-3691 (2013).
  • Halberg F., Powell D., Otsuka K., Watanabe Y., Beaty L.A., Rosch P., Czaplicki J., Hillman D., Schwartzkopff O. and Cornelissen G. Diagnosing vascular variability anomalies, not only MESOR-hypertension, Am J Physiol Heart Circ Physiol. 305, 279-94 (2013).

2012

  • Iordanov, I., Renault, M., Reat, V., Bosshart, P. D., Engel, A., Saurel, O., and Milon, A. Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: The four extracellular loops display restricted motion behavior in micelles and in lipid bilayers, Biochim Biophys Acta 1818, 2344-2353 (2012).
  • Bosshart, P. D., Iordanov, I., Garzon-Coral, C., Demange, P., Engel, A., Milon, A., and Muller, D. J. The Transmembrane Protein KpOmpA Anchoring the Outer Membrane of Klebsiella pneumoniae Unfolds and Refolds in Response to Tensile Load, Structure 20, 121-127 (2012).
  • Campagne, S., Muller, I., Milon, A., and Gervais, V. Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1, Nucleic Acids Res 40, 9927-9940 (2012).
  • Arab-Jaziri, F., Bissaro, B., Barbe, S., Saurel, O., Debat, H., Dumon, C., Gervais, V., Milon, A., Andre, I., Faure, R., and O’Donohue, M. J. Functional roles of H98 and W99 and beta2alpha2 loop dynamics in the alpha-l-arabinofuranosidase from Thermobacillus xylanilyticus, FEBS J 279, 3598-3611 (2012).
  • Mazarguil, H., Mollereau, C., Czaplicki, G., and Zajac, J. M. Study of the N-terminal part of peptidic selective NPFF2 agonists, Peptides 37, 157-160 (2012).
  • Renault, M., Pawsey, S., Bos, M. P., Koers, E. J., Nand, D., Tommassen-van Boxtel, R., Rosay, M., Tommassen, J., Maas, W. E., and Baldus, M. Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization, Angew Chem Int Edit 51, 2998-3001 (2012).
  • Renault, M., Tommassen-van Boxtel, R., Bos, M. P., Post, J. A., Tommassen, J., and Baldus, M. Cellular solid-state nuclear magnetic resonance spectroscopy, Proc Natl Acad Sci USA 109, 4863-4868 (2012).

2011

  • Rath, P., Demange, P., Saurel, O., Tropis, M., Daffe, M., Dotsch, V., Ghazi, A., Bernhard, F., and Milon, A. Functional expression of the PorAH channel from Corynebacterium glutamicum in cell-free expression systems: implications for the role of the naturally occurring mycolic acid modification, J Biol Chem 286, 32525-32532 (2011).
  • Irague, R., Massou, S., Moulis, C., Saurel, O., Milon, A., Monsan, P., Remaud-Simeon, M., Portais, J. C., and Potocki-Veronese, G. NMR-based structural glycomics for high-throughput screening of carbohydrate-active enzyme specificity, Anal Chem 83, 1202-1206 (2011).
  • Demange, P., Réat, V., Weinandy, S., Ospital, R., Chopinet-Mayeux, L., Henri, P., Milon, A., and Teissié, J. Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells, J. Biomater. Nanobiotech. 2, 527-532 (2011).
  • Campagne, S., Gervais, V., and Milon, A. Nuclear magnetic resonance analysis of protein-DNA interactions,J R Soc Interface 8, 1065-1078 (2011).
  • Renault, M., Bos, M. P., Tommassen, J., and Baldus, M. Solid-State NMR on a Large Multidomain Integral Membrane Protein: The Outer Membrane Protein Assembly Factor BamA, J Am Chem Soc 133, 4175-4177 (2011).

Download the complete list of publications from 2016 to 2002

Collaborations inside France

  • L. Mourey, M. Daffé, M. Tropis, P. Calsou, O. Cuvillier, IPBS, University of Toulouse – CNRS
  • M. Remaud-Siméon, A. Marty, I. André, R. Fauré, M. J. O’Donohue, LISBP, University of Toulouse – INRA - CNRS
  • J. F. Arnal, I2MC, University of Toulouse – INSERM
  • J.L. Banères, IBMM, Montpellier
  • S. Granier, IGF, Montpellier
  • G. Pintacuda, CRMN, Lyon

 

International Collaborations

  • M. Baldus, Utrecht University, The Netherlands
  • L. Eggeling, IBG-1, Jülich, Germany
  • V. Cherezov and R. Stevens, USC, Los Angeles, USA
  • K. Wüthrich, The Scripps Research Institute, La Jolla, USA
  • J.A. Bengoechea, Queen’s University, Belfast, UK