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Mieux comprendre les bases de la reconnaissance moléculaire du vivant, d'un point de vue fondamental et biomédical, voilà l'intérêt primordial de la recherche menée par Bruno Klaholz et son équipe. Après des études en chimie à Karlsruhe (Allemagne) il s'oriente vers la biochimie et la cristallographie aux rayons-X à Strasbourg afin d'étudier les relations structure-fonction de ligands liés aux récepteurs d'hormones, d'importantes cibles en pharmacologie. Puis, afin d'appréhender l'architecture tridimensionnelle de complexes macromoléculaires de grande taille - voire dans le contexte cellulaire -, il s'intéresse aux techniques de traitement d'image et reconstruction 3D par cryo microscopie électronique au cours de son post-doctorat à Londres (Imperial College), avant de monter à Strasbourg une équipe de recherche s'intéressant aux grand complexes impliqués dans la régulation des gènes. Les études sur le ribosome (complexes d'initiation et de terminaison de la traduction, polyribosomes, première structure du ribosome humain) et des complexes de transcription (récepteurs nucléaires, complexes chromatiniens) sont menées dans l'esprit d'une intégration multi-échelle incluant notamment biochimie, bio-informatique, cristallographie aux rayons-X, cryo microscopie électronique à haute résolution, cryo tomographie électronique et microscopie à fluorescence super-résolutive, participant ainsi activement aux développements menés au Centre de Biologie Intégrative (CBI) dans le cadre des infrastructures nationales et européennes. |
Bruno Klaholz
Centre for Integrative Biology (CBI), Department of Integrated Structural Biology, IGBMC (Institute of Genetics and of Molecular and Cellular Biology), 67400 Illkirch, France.
Integrated Structure Analysis of Nucleoprotein Complexes in Action
Large nucleoprotein complexes are challenging biological objects to study and require an integrated structure-function approach. The latter allows bridging various scales and resolution levels, from the atomic to the cellular level (Ménétret et al., 2013; Urzhumtseva et al., 2013, Orlov et al., 2015, Beinsteiner et al., Andronov et al.). In an effort to favour synergies across complementary disciplines we are combining biochemistry, bioinformatics, X-ray crystallography, single particle cryo electron microscopy (cryo-EM), cryo electron tomography (cryo-ET) and correlative fluorescence imaging in order to address the structure-function relationships of complexes involved in gene expression regulation, with a focus on ribosome complexes (Simonetti et al., 2013a & 2013b) and human DNA-bound nuclear receptors (Maletta et al., Nat. Com. 2014). The presentation will focus on the integrated structural biology approach that we developed for the study of the human ribosome for which obtaining an atomic structure represented a major challenge to address. This included biochemistry, biophysical characterization with MALLS, AUC, MS and fluorescence anisotropy, single particle cryo-EM visualization for sample characterization and optimization of the sample preparation protocol and X-ray crystallography, which altogether lead to first, but weakly diffracting crystals of the human 80S ribosome (Khatter et al., 2014). From these optimized samples, we determined the near-atomic structure of the human ribosome using high-resolution single particle cryo electron microscopy using the in-house Titan Krios electron microscope, advanced image processing and dedicated computing resources, and atomic model building using new crystallography refinement procedures. The structure reaches 2.9 Å resolution in the most stable regions and thus provides unprecedented insights into rRNA entities and amino acid side-chains of the human ribosome (Khatter et al., 2015). Furthermore, we will describe the 3D organization of ribosomes when bound to an mRNA strand and forming poly-ribosome assemblies such as left-handed supramolecular helices which we analysed by single and dual-axis cryo electron tomography (Myasnikov et al., 2013; Afonina et al., 2014; Afonina et al., 2015; Myasnikov et al., 2014). Finally, an outlook will be given on ongoing developments in super-resolution fluorescence microscopy applied to chromatin complexes. Together, these results highlight the importance of advanced technologies in structural biology (high-resolution cryo electron microscopes, sensitive direct electron detectors, super-resolution imaging and X-ray crystallography, including structure sorting, Klaholz 2015) to allow a better multi-scale multi-resolution integration, notably for correlative microscopy at the light and electron microscopy interfaces. Such integrated tools are available through project-based access through the European and French distributed infrastructures http://frisbi.eu/ and http://www.structuralbiology.eu/.
References
[1] J-F. Ménétret, H. Khatter, A. Simonetti, I. Orlov, A. G. Myasnikov, Vidhya KV, S. Manicka, M. Torchy, K. Mohideen, A-S. Humm, I. Hazemann, A. Urzhumtsev, B. P. Klaholz. Integrative structure-function analysis of large nucleoprotein complexes. RNA structure and folding, de Gruyter, 2013, D. Klostermeier & C. Hammann (Eds.).
[2] Simonetti A, Marzi S, Billas IM, Tsai A, Fabbretti A, Myasnikov AG, Roblin P, Vaiana AC, Hazemann I, Eiler D, Steitz TA, Puglisi JD, Gualerzi CO, Klaholz BP. Involvement of protein IF2 N domain in ribosomal subunit joining revealed from architecture and function of the full-length initiation factor. Proc Natl Acad Sci U S A, 2013; 110:15656-61.
[3] Simonetti A, Marzi S, Fabbretti A, Hazemann I, Jenner L, Urzhumtsev A, Gualerzi CO, Klaholz BP. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms. Acta Crystallogr D, 2013; 69:0925-33.
[4] Myasnikov AG, Afonina ZA, Klaholz BP. Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography. Ultramicroscopy 2013; 126:33-9.
[5] Urzhumtseva L, Klaholz B, Urzhumtsev A. On effective and optical resolutions of diffraction data sets. Acta Crystallogr D, 2013.
[6] Afonina Z. A., A. G. Myasnikov, V. A. Shirokov, B. P. Klaholz, A. S. Spirin. Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study. Nucleic Acids Res. 2014, 42, 9461-9. doi: 10.1093/nar/gku599.
[7] Myasnikov A. G., Z. A. Afonina, J-F. Ménétret, V. A. Shirokov, A. S. Spirin & B. P. Klaholz. The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes. Nat Commun., 2014, 5, 5294. doi: 10.1038/ncomms6294.
[8] Afonina Z. A., A. G. Myasnikov, V. A. Shirokov, B. P. Klaholz, A. S. Spirin. Conformation transitions of eukaryotic polyribosomes during multi-round translation. Nucleic Acids Res. 2015, 43, 618-28. doi: 10.1093/nar/gku1270.
[9] Maletta, I. Orlov, P. Roblin, Y. Beck, D. Moras, I. M. L. Billas & B. P. Klaholz. The palindromic DNA-bound USP/EcR nuclear receptor adopts an asymmetric organization with allosteric domain positioning. Nat Commun., 2014, 5, 4139. doi: 10.1038/ncomms5139.
[10] I. Orlov, A. Schertel, G. Zuber, B. P. Klaholz, R. Drillien, E. Weiss, P. Schultz & D. Spehner. Live cell immunogold labelling of RNA polymerase II. Sci. Rep. 2015, 5, 8324.
[11] Khatter H, Myasnikov AG, Mastio L, Billas IM, Birck C, Stella S, Klaholz BP. Purification, characterization and crystallization of the human 80S ribosome. Nucleic Acids Res., 2014, 42(6), 1-11.
[12] H. Khatter, A. G. Myasnikov, K. Natchiar & B. P. Klaholz. Structure of the human 80S ribosome. Nature, 2015, 520, 640-5. doi: 10.1038/nature14427.
[13] B. Beinsteiner, J. Michalon & B. P. Klaholz. IBiSS, a versatile and interactive tool for integrated sequence and 3D structure analysis of large macromolecular complexes. Bioinformatics, 2015. pii: btv347.
[14] B. P. Klaholz. Structure sorting of multiple macromolecular states in heterogeneous cryo-EM samples by 3D multivariate statistical analysis. Open Journal of Statistics, 2015, 5, 820-836.
[15] L. Andronov, Y. Lutz, J-L. Vonesch & B. P. Klaholz. ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy. Submitted.
[16] L. Andronov, Y. Lutz, J-L. Vonesch & B. P. Klaholz. SharpViSu: integrated analysis of super-resolution microscopy data. Submitted. |
