Modulation of Ndc80-microtubule interactions by supramolecular ligands and physiological regulators

The eukaryotic cell cycle depends on the interplay between many protein complexes that ultimately ensure the precise duplication and the accurate segregation of the genome during cell division. In mitosis, the spindle attaches to the chromosomes via large protein complexes called kinetochores. In anaphase, spindle microtubules depolymerize and pull the sister chromatids apart. The direct contact between the kinetochore and the microtubule is mediated by the calponin homology (CH) domain of the conserved Ndc80 complex. Here, multiple surface-exposed lysines are essential for binding to the microtubule surface. In addition, phosphorylation of the N-terminal Ndc80 segment preceding the CH-domain by the mitotic kinases Ipl1 and Mps1 is thought to regulate microtubule binding. This is crucial during mitotic error correction when sister chromatids gradually achieve bi-oriented attachments. Due to its importance for a fundamental cellular mechanism, a number of chemical compounds targeting Ndc80 have been described, however many of them seem to predominantly impair microtubule dynamics instead of targeting the Ndc80 CH-domain directly. In this work, the yeast Ndc80 CH-domain was introduced as a model protein to develop new supramolecular ligands and to investigate regulatory mechanisms. The 15 kDa protein was shown to have a compact globular, highly α-helical structure, that could bind microtubules depending on the presence of critical lysine residues at its surface. Starting from the established Lys/Arg-directed monomeric molecular tweezer CLR01, multivalent tweezer assemblies, varying in number of binding moieties, as well as length and structure of backbone connections, were developed and tested as ligands of the CH-domain. High affinity binding was established for a new set of multivalent tweezers, whereas the monomeric prototype, CLR01, was considerably less potent. The new supramolecular ligands inhibited binding of the full-length Ndc80 complex to microtubules in a dose-dependent manner, as judged by TIRF microscopy. Tubulin polymerization was not impaired in the concentration window sufficient for inhibition of microtubule-binding. NMR titrations, supported by in silico modelling and simulations localized the interactions sites of the tweezers to K152/160 and K204/217. The linker-arms of the multimeric tweezer assemblies played an important role in the binding mechanism as they provided flexibility to adapt to the CH-domain surface, allowing binding of multiple lysines simultaneously. In the second part of this work, the influence of the N-terminal segment on CH-domain function and regulation was investigated. To this end N-CH constructs, including mutations that mimic the effects of the kinases Ipl1 and Mps1, were purified and characterized. As expected, NMR experiments identified the N-tail as disordered in comparison to the compact CH-domain. Phosphomimetic mutations inhibited microtubule binding to varying degrees and altered intramolecular chemical crosslinking patterns of the constructs. Surprisingly, wildtype N-CH not only bound microtubules, but also soluble tubulin, a feature not previously documented for Ndc80. Combination of the wildtype N-CH with tubulin at high concentrations led to the formation of small stub-like filaments, reminiscent of increased microtubule nucleation. Chemical crosslinking revealed contacts between N-CH predominantly with α-tubulin, and phosphomimetic variants displayed fewer crosslinks. Regulated interactions of Ndc80 with tubulin dimers may influence polymerization dynamics at the kinetochore, an idea that should be investigated in future experiments.