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bioRxiv · 10.1101/2022.03.31.486303

Quantitative crosslinking and mass spectrometry determine binding interfaces and affinities mediating kinetochore stabilization

Hagemann, G.·Solis-Mezarino, V.·Singh, S.·Potocnjak, M.·Kumar, C.·Herzog, F.
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Abstract

Crosslinking and mass spectrometry (XLMS) are used in integrative structural biology to acquire spatial restraints. We found a dependency between crosslink distances and intensities and developed a quantitative workflow to simultaneously estimate apparent dissociation constants (KD) of contacts within multi-subunit complexes and to aid interface prediction. Quantitative XLMS was applied to study the assembly of the macromolecular kinetochore complex, which is built on centromeric chromatin and establishes a stable link to spindle microtubules in order to segregate chromosomes during cell division. Inter-protein crosslink intensities facilitated determination of phosphorylation-induced binding interfaces and affinity changes. Phosphorylation of outer and inner kinetochore proteins mediated cooperative kinetochore stabilization and decreased the KD values of its interactions to the centromeric nucleosome by ~200-fold, which was essential for cell viability. This work demonstrates the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modeling.

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Hagemann, G., Solis-Mezarino, V., Singh, S., Potocnjak, M., Kumar, C., Herzog, F.. 2022-04-01. Quantitative crosslinking and mass spectrometry determine binding interfaces and affinities mediating kinetochore stabilization. https://doi.org/10.1101/2022.03.31.486303

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