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

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.