End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells and purified systems, the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates spontaneous nucleation and growth of microtubules, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets, which rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule-sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing-ends together with the elimination of stressed microtubule lattices may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues.

Background information. Hsp90 (90 kDa heat-shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide-binding-dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue). Results and discussion.Here, we present the first nucleotide-free structures of the entire eukaryotic Hsp90 (apoHsp90) obtained by small-angle X-ray scattering and single-particle cryo-EM (cryo-electron microscopy). We show that, in solution, apo-Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo-EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo-forms require large movements of the NTD (N-terminal domain) and MD (middle domain) around two flexible hinge regions.Conclusions. The present study shows, for the first time, the structure of the entire eukaryotic apo-Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.

Aggregation of TDP-43 (transactive response DNA binding protein 43 kDa) is a hallmark of certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, intracellular TDP-43-positive inclusions are often found in other neurodegenerative diseases. Recently it was shown that zinc ions can provoke the aggregation of endogenous TDP-43 in cells, allowing to assume a direct interaction of TDP-43 with zinc ions. In this work, we investigated zinc binding to the 102–269 TDP-43 fragment, which comprise the two RNA recognition motifs. Using isothermal titration calorimetry, mass spectrometry, and differential scanning fluorimetry, we showed that zinc binds to this TDP-43 domain with a dissociation constant in the micromolar range and modifies its tertiary structure leading to a decrease of its thermostability. Moreover, the study by dynamic light scattering and negative stain electron microscopy demonstrated that zinc ions induce auto-association process of this TDP-43 fragment into rope-like structures. These structures are thioflavin-T-positive allowing to hypothesize the direct implication of zinc ions in pathological aggregation of TDP-43.