Interaction de la tubuline avec des composés qui régulent son assemblage en microtubules

Les microtubules (MTs), composants clés du cytosquelette des cellules eucaryotes, sont des tubes constitués d hétérodimères de tubuline. La structure de la tubuline complexée au domaine stathmine de la protéine RB3 (complexe T2R) et à la colchicine a été déterminée précédemment par cristallographie aux rayons X et constitue le point de départ de ce travail. Dans le but d améliorer la qualité des cristaux de T2R, notre étude a contribué à mieux contrôler l ensemble du processus qui permet d étudier l interaction de la tubuline avec des ligands d intérêt, de la purification de RB3 à la cristallisation de T2R. Les résultats ouvrent la voie à des recherches futures pour obtenir d autres formes cristallines de tubuline. Nous avons aussi déterminé la structure de la tubuline dans T2R avec trois ligands du site colchicine. Ces données définissent le cadre structural de la conception rationnelle de molécules à visée thérapeutique se fixant au même site de la tubuline et inhibant son assemblage.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Studying Drug-Tubulin Interactions by X-Ray Crystallography

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Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

International audienceStructural changes occur in the alphabeta-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a beta-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with alpha-tubulin from stacking onto a beta-tubulin beta sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the beta-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

International audienceThe initiation of mRNA degradation often requires deprotection of its 5′ end. In eukaryotes, the 5′-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5′-monophosphorylated RNA that is a substrate for 5′ exoribonucleases. In bacteria, the 5′-triphosphate group of primary transcripts is also converted to a 5′ monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5′ exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNAtargets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria