Abstract
Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1-6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.
Highlights
Basic cellular homeostasis depends on the regulated protein degradation and turnover by the ubiquitin-proteasome system [1, 2]
Pair-wise subunit interactions derived from the identified disuccinimidyl sulfoxide (DSSO) cross-linked peptides were the basis for subsequent probabilistic analysis to obtain the spatial organization of the 19S regulatory particle (RP) subcomplexes
We have successfully employed a new integrated crosslinking strategy to unravel the spatial organization of the yeast 19S RP complex through the identification of cross-linked peptides and probabilistic modeling
Summary
Basic cellular homeostasis depends on the regulated protein degradation and turnover by the ubiquitin-proteasome system [1, 2]. To facilitate MS detection and identification of cross-linked products, we have recently developed a novel homobifunctional amine reactive, low energy MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO), and successfully applied it to cross-link the yeast 20S proteasome for rapid, accurate, and simplified determination of protein interaction interfaces within the complex [25]. Current cross-linking studies have been focused on protein complexes with known crystal structures, but topological structures of protein complexes based primarily on cross-linking data have not yet been reported. The resulting cross-linking information serves as a basis for a rigorous probabilistic analysis to obtain the maximum likelihood (ML) topology This strategy is developed by first analyzing our cross-linking data for the 19S six-member AAA-ATPase base ring, as the topology ordering of yeast orthologs has been recently determined (14, 26 –28). This work represents the first application of probabilistic modeling of protein complexes based solely on cross-link data, establishing a new workflow for future structural analysis of large protein complexes using XL-MS
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