Abstract
Cells organize their actions partly through tightly controlled protein-protein interactions-collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the protein-protein interactions in intact human nuclei. Overall, we identified ∼8,700 crosslinks, of which 2/3 represent links connecting distinct proteins. From these data, we gain insights on interactions involving histone proteins. We observed that core histones on the nucleosomes expose well-defined interaction hot spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build low-resolution models of their binding mode to the nucleosome. For HMGN2, the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS, and modeling. Excitingly, the analysis of crosslinks carrying posttranslational modifications allowed us to extract how specific modifications influence nucleosome interactions. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.
Highlights
Cells organize their actions partly through tightly controlled protein-protein interactions—collectively termed the interactome
A Crosslinking Strategy Preserving Endogenous Nuclear Protein-Protein Interactions—We hypothesized that the large transporters, such as the nuclear pore complexes, embedded in the nuclear envelope could expedite diffusion of the crosslinking reagent disuccinimidyl sulfoxide (DSSO) into the nucleus, enabling crosslinking of the nuclear proteins in their natural environment (Fig. 1A)
From the acquired mass spectrometry data of both the soluble as well as the insoluble fractions, we identified 7,095 unique crosslinks at an false discovery rate (FDR) of 1% (Fig. 1C; supplemental Table S2)
Summary
Cells organize their actions partly through tightly controlled protein-protein interactions—collectively termed the interactome. To extract detailed insights in the nucleosome PPIs, we generated a Lys-reactivity map to highlight the residues on the core histones that were involved in interprotein crosslinks (Fig. 3A).
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