Study of nuclear hormone receptors by an integrative structural biology approach. – NRcoactiv

1-Scientific background and objectives: Eukaryotic transcription is a fundamental mechanism of gene activity regulation and has profound implications in human health. The ligand-dependent transcription regulation by nuclear receptors involves the transient assembly of large macromolecular complexes. The complexes formed by the nuclear receptors bound to DNA response elements recruit co-activation protein complexes in order to facilitate the assembly of the general transcription machinery on the promoter of the target gene. The aim of the present project is to take up the challenge of determining the architecture of an entire nuclear receptor, and to reveal the functional and structural determinants of ligand-dependent transcription activation by co-activator proteins.

2-Description of the project, methodology: The study of macromolecular complexes involved in transcription requires an integrative approach including molecular biology, bio-physics, bio-informatics and structural biology. The latter comprises the combination of different, yet complementary techniques, in particular cryo-electron microscopy (cryo-EM) and 3-D reconstruction of single molecules, small-angle X-ray diffraction (SAXS) of complexes in solution, and crystallography for detailed insights into protein-protein and protein-DNA interactions and their implications in the ligand-dependent molecular mechanism. Making use of the in-house technical platforms for cloning and crystallisation, we plan the purification, characterisation and structural study of complexes of either human or insect nuclear receptors with their respective co-activator proteins, taking advantage of the simplified transcription model system in insects. The structural study by cryo-EM will use state-of-the-art techniques developed in the coordinator's team, in particular for image processing and 3-D reconstruction of the complexes. The feasibility of the project is supported by two recent breakthroughs at the IGBMC concerning the purification of a full-length nuclear receptor and the cryo-EM study of this complex. In order to optimise the detailed interpretation, the resolution of the cryo-EM structures will be pushed to the limits by processing large data sets.

3-Expected results: The dynamics of the macromolecular complexes and the structural conformations of the co-activation complexes will be addressed based on molecular models obtained by precisely fitting the crystal structures of the individual nuclear receptor domains into high-resolution cryo-EM structures. Thus, the structural and functional study of the co-activation complexes will allow revealing the architecture of the entire functional unit formed by nuclear receptors bound to different response elements and to evaluate the effects of therapeutic ligands (synthesized and provided by industrial partners) on the molecular mechanism of transcription activation.