Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome

Abstract

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based and XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…Until now, only a limited number of in vivo XL-MS studies have been reported, mainly due to the challenges discussed above. While the Bruce lab has focused on in vivo cross-linking at the proteome level using their PIR cross-linkers 40,77,89,104–106,108,139,151 , our lab has mostly focused on in vivo XL-MS analysis of protein complexes 79,99,159,162,166,167 . Recently, we have developed a membrane permeable, sulfoxide-containing MS-cleavable and enrichable NHS ester cross-linker Azide-A-DSBSO (Table 2) 79 .…”

“…With the advancement of cryo-EM and hybrid structural tools, high-resolution structures of 26S proteasomes have been determined in recent years 33,202–205 . Through this process, XL-MS analysis has contributed significantly to the elucidation of proteasome architectures and to uncovering structural details underlying proteasome function and regulation 23,33,95,98,99,206–209 . In an early study, XL-MS and EM were used to define the structural topology of the AAA-ATPase module in the S.pombe 19S RP, as well as its spatial relationship to the α-ring of the 20S core particle 206 .…”

“…These results suggest conformational heterogeneity of the human 26S proteasome. Apart from the 26S holocomplex, additional cross-links were identified to demonstrate direct physical interactions between proteasome subunits and 15 proteasome-interacting proteins, including 9 known and 6 new interactors 99 . Among them, UBLCP1, is the only known proteasome phosphatase 211 , whose dynamic interaction with Rpn1 has been confirmed by XL-MS and integrative modeling with a proposed model inferring the action modes of UBLCP1 on regulating proteasomes.…”

An integrated workflow for crosslinking mass spectrometry

“…The technological development currently focuses on enrichment strategies for crosslinked peptides and mass spectrometric data acquisition (Leitner et al , ; Kolbowski et al , ; Liu et al , ), including newly designed crosslinkers (Kao et al , ). MS2‐cleavable crosslinkers, in particular, have celebrated recent successes for the analysis of protein complexes (Wang et al , ) or complex mixtures (Chavez et al , ; Liu & Heck, ).…”

Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein–Protein Interactions

“…These unique characteristics enable straightforward and unambiguous identification of cross-linked peptides by MS n analysis coupled with conventional database searching tools 9,17,19,20 . Sulfoxide-containing MS-cleavable cross-linkers have been successfully applied to not only study PPIs in vitro 17,24–30 and in vivo 20,27 , but also to quantify structural dynamics of protein complexes 19,31 . Given the robustness of sulfoxide-based cleavability, we aimed to design, synthesize and characterize a novel sulfoxide-containing MS-cleavable homobifunctional cysteine linker, namely, BMSO ( B is- m aleimide s ulf o xide) to facilitate the identification of cysteine cross-linked peptides.…”