Author:
Hagemann Goetz,Solis-Mezarino Victor,Singh Sylvia,Potocnjak Mia,Kumar Chandni,Herzog Franz
Abstract
AbstractCrosslinking 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.
Publisher
Cold Spring Harbor Laboratory
Cited by
3 articles.
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