title: 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-basedin vivoandin vitroXL-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.
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, Irvine, California 92697.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.affiliation: §Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, California 94143.
authors.forename: Peter
authors.initials: P
authors.lastname: Cimermancic
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, Irvine, California 92697.
authors.forename: Clinton
authors.initials: C
authors.lastname: Yu
authors.affiliation: ¶Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schweitzer
authors.affiliation: §Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, California 94143.
authors.forename: Nikita
authors.initials: N
authors.lastname: Chopra
authors.affiliation: ‖Department of Pharmacology, University of California, San Diego, La Jolla, California, 92093.
authors.forename: James L
authors.initials: JL
authors.lastname: Engel
authors.affiliation: §Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, California 94143.
authors.forename: Charles
authors.initials: C
authors.lastname: Greenberg
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, Irvine, California 92697.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Huszagh
authors.affiliation: ¶Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Beck
authors.affiliation: ¶Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Eri
authors.initials: E
authors.lastname: Sakata
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, Irvine, California 92697.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Yang
authors.affiliation: **Department of Chemistry, University of California, Irvine, Irvine, California 92697.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Novitsky
authors.affiliation: ‡‡Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Leitner
authors.affiliation: §§Functional Genomics Center Zurich (FGCZ), University of Zurich, ETH Zurich, CH-8057 Zurich, Switzerland.
authors.forename: Paolo
authors.initials: P
authors.lastname: Nanni
authors.affiliation: ¶¶Institute of Molecular Life Sciences, University of Zurich, CH-8057 Zurich, Switzerland.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Kahraman
authors.affiliation: ‖Department of Pharmacology, University of California, San Diego, La Jolla, California, 92093.
authors.forename: Xing
authors.initials: X
authors.lastname: Guo
authors.affiliation: ‖Department of Pharmacology, University of California, San Diego, La Jolla, California, 92093.
authors.forename: Jack E
authors.initials: JE
authors.lastname: Dixon
authors.affiliation: **Department of Chemistry, University of California, Irvine, Irvine, California 92697.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Rychnovsky
authors.affiliation: ‡‡Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, Zurich, Switzerland.|Faculty of Science, University of Zurich, Zurich, Switzerland.
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.affiliation: ¶Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried 82152, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.affiliation: §Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, California 94143.
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
authors.affiliation: From the ‡Department of Physiology & Biophysics, University of California, Irvine, Irvine, California 92697; lanhuang@uci.edu.
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
mesh_terms: D002460:Cell Line; D006801:Humans; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease