KINETOCHORE MAP · Analysis of the kinetochore architecture and dynamics using a mass spectrometry based integrative structural approach

Analysis of the kinetochore architecture and dynamics using a mass spectrometry based integrative structural approach

Summary The goal of this project is to establish an integrative mass spectrometric approach to analyze the structure of multi-subunit protein complexes. Structural information is obtained in form of distance restraints by combining chemical cross-linking and mass spectrometry (XL-MS). The distance information is sufficient to integrate subunit X-ray structures and electron microscopic (EM) density maps by computational modeling. This type of hybrid structural biology analysis is an emerging approach tailored to the structural investigation of macromolecular protein complexes which are often flexible and heterogeneous and therefore not amenable to established structural biology techniques. To understand the assembly of protein complexes we determine the copy number of subunits by label free quantification using mass spectrometry. Information about the relative quantities of subunits reveals its subunit stoichiometry which is a prerequisite for elucidating the architecture of protein complexes. We further estimate the total copy numbers of complex subunits in a cell which is important for gaining insights into protein complex assembly. This structural proteomics approach will be applied to elucidate the topology of kinetochore complexes and the protein phosphatise 2A (PP2A) network which are key protein complexes regulating the faithful dissemination of sister chromatids during mitosis and cell cycle progression. To establish a robust XL-MS protocol applicable to various types of protein samples we optimized the different steps of the workflow. Improving the cross-linking reaction, the enrichment of the cross-linked peptides by size exclusion and their mass spectrometric detection as well as a modified strategy to identify the cross-linked by the search engine xQuest substantially increased the quality and the yield of the obtained distance restraints. The improved protocol was first tested by analyzing protein complexes purified for crystallographic purposes. The high purity and concentration of these complexes facilitated the validation and further optimization of our XL-MS protocol on macromolecular complexes. In collaboration with the laboratory of Patrick Cramer we investigated the structure of RNA polymerase I and III purified from Saccharomyces cerevisiae. The subunit topology of RNA polymerase I and III was revealed by 150 and 100 inter-protein cross-links, respectively. Based on the X-ray structure of the 12-subunit core of RNA polymerase II this analysis showed that about 95% of inter-protein cross-links were detected between physically interacting subunits. Inter-protein cross-links thus indicate direct protein-protein interaction. Inter-subunit distance restraints clarified the transient association of the transcription initiation factor Rrn3. Furthermore, the XL-MS analysis provided functional insights elucidating the structural basis of RNA polymerase I intrinsic 3’ RNA cleavage activity that is implicated in proofreading and transcription termination. In a next step, I developed a strategy to identify cross-link restraints on protein complexes affinity-purified from human tissue culture cells. As these protein preparations are heterogeneous and at low concentration a second affinity-step was used for re-concentration and buffer exchange. Protein complexes of the kinetochore and the PP2A network were purified form HEK293 cells by inducibly expressing an affinity-tagged subunit. The identification of about 30 inter-protein cross-links each on the outer kinetochore Ndc80 and Mis12 complexes were sufficient to describe their subunit topology and were consistent with recently published structures of these complexes. Reciprocal affinity-purifications of 14 proteins resulted in a PP2A high-density interaction network of 81 proteins displaying 256 interactions. Application of our XL-MS workflow on protein complexes isolated from tissue culture cells yielded 180 inter-protein and 560 intra-protein distance restraints that link specific trimeric PP2A complexes to a multitude of adaptor proteins controlling its cellular function. Distance restraints advanced an interaction network of co-purifying proteins to a topological map. This is the first comprehensive study revealing direct protein-protein interactions within a signaling pathway. These distance restraints will be used in hybrid structural biology approaches and are expected to significantly improve computational molecular modeling efforts that provide detailed structural insights into protein complexes assembling the PP2A signaling network. To date, systematic estimates of cellular protein concentrations are exceptionally scarce. The generation of mathematical models of biological processes, the simulation of these processes under different conditions, and the comparison and integration of multiple data sets are explicit goals of systems biology that require the knowledge of the absolute quantity of the system's components. Using the previously described directed mass spectrometry approach, we quantified the proteome of the commonly used human cell line U2OS in two functional states, interphase and mitosis. We showed that these human cultured cells express at least ~10 000 proteins and that the quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell. We demonstrated that cellular core functions are often carried out by relatively few proteins, which are present at very high abundance in the cell. In contrast, regulatory functions are often orchestrated by large protein families existing in variable but predominantly low abundance. The method used in this study is principally applicable to the majority of all cell types and might be useful to investigate a multitude of cellular states and organisms in the future. The determination of the total copy number of protein complex subunits in a cell is a prerequisite to understand the participation of a certain protein in various different complexes. Investigating the dynamics of protein abundances between different cellular states will be crucial for gaining insights into the timely assembly of key protein complexes controlling cell cycle progression.

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