Quantitative crosslinking and mass spectrometry indicates kinetochore complex stabilization
Goetz Hagemann1, Victor Solis-Mezarino1, Sylvia Singh1, Mia Potocnjak1, Chandni Kumar1, Benjamin Neuditschko2 and Franz Herzog1,2,*
1 Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany
2 Institute Krems Bioanalytics, IMC University of Applied Scienes Krems, 3500 Krems, Austria
Introduction: Distance restraints derived from the mass spectrometric identification of crosslinked amino acids (XLMS) are widely applied in integrative approaches to determine protein connectivity and to model the topology of protein complexes. Besides structure, the critical determinant of the molecular mechanism of a complex is the interaction strength of its subunit contacts, which can be modulated through cofactors or post-translational modifications. We reasoned that crosslink intensities provide a quantitative measure for the formed complex and the free subunits at the equilibrium state. Thus, we investigated whether crosslink intensities facilitate the simultaneous estimation of individual protein-protein affinities within kinetochore multi[1]subunit complexes.
Methods: Protein complexes were crosslinked by modifying the α-amino groups with the isotopically labeled BS2G-d0/d6 reagent and crosslinked peptide fractions were analyzed by liquid chromatography coupled to tandem mass spectrometry. The raw files were processed by the xQuest/xProphet software to identify the crosslinked peptides, their precursor ion masses and retention times. This information was subsequently used for the extraction of ion chromatograms by the OpenMS software tool. We further applied inter- and intra-protein protein crosslink intensities to estimate the concentrations of the formed complex and the free subunits according to the steady state equilibrium in solution. To assess whether crosslink intensities supported the estimation of binding affinities and interfaces we titrated recombinant kinetochore subunits for complex formation over a range of molar ratios.
Preliminary data: We found a dependence between crosslink distances and intensities and applied a quantitative workflow to estimate binding affinities and aid interface prediction of kinetochore subunit contacts which link chromosomes to spindle microtubules. Titrating the assembly of 11 subunits showed that phosphorylation induces a high-affinity link to the centromeric nucleosome required for transmitting forces of depolymerizing microtubules. Phosphorylation of Mif2CENP-C putatively by Cdc5PLK1 induced binding to Ame1/Okp1CENP-U/Q in vitro and mediated their cooperative stabilization at the Cse4CENP-A nucleosomes which was enhanced by binding of the MTW1MIS12 complex phosphorylated by Ipl1Aurora-B. Together, both phosphorylation events decreased the KD-values of Cse4CENP-A interactions by ~300-fold and are essential for cell viability. This work demonstrates the potential of quantitative XLMS for characterizing the stabilization of the kinetochore at the centromeric nucleosome through phosphorylation of the outer and inner kinetochore in mitosis.
Novel aspect: 1. Crosslink intensities as quantitative measures for the formed complex and the free subunits at equilibrium allow estimation of binding affinities in multi-subunit complexes. 2. Correlation between increasing crosslink intensity and decreasing distance to protein contact facilitates narrowing down binding interfaces
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