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

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 in vivo and in vitro 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. 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 in vivo and in vitro 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. The ubiquitin-proteasome system (UPS) 1The abbreviations used are: UPS, ubiquitin proteasome system;XL-MS, cross-linking mass spectrometry;CP, core particle;RP, regulatory particle;UBL, ubiquitin-like domain;UBA, ubiquitin associating domain;AP-MS, affinity purification mass spectrometry;PIP, proteasome interacting proteins;HB, histidine and biotin tag;LC MSn, liquid chromatography multistage tandem mass spectrometry. 1The abbreviations used are: UPS, ubiquitin proteasome system;XL-MS, cross-linking mass spectrometry;CP, core particle;RP, regulatory particle;UBL, ubiquitin-like domain;UBA, ubiquitin associating domain;AP-MS, affinity purification mass spectrometry;PIP, proteasome interacting proteins;HB, histidine and biotin tag;LC MSn, liquid chromatography multistage tandem mass spectrometry.represents the major intracellular pathway for selective removal of regulatory, misfolded, and damaged proteins in eukaryotic cells. Aberrant UPS regulation can result in irregular protein turnover and accumulation of dysfunctional proteins, thus leading to various human diseases. The 26S proteasome is the macromolecular machine in the UPS that is responsible for controlled degradation of ubiquitinated substrates (1.Voges D. Zwickl P. Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis.Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1595) Google Scholar). It is composed of at least 33 subunits, which assemble into two subcomplexes: the 20S core particle (CP) and the 19S regulatory particle (RP). The 20S CP is responsible for various proteolytic activities, and has a highly conserved "barrel"-like structure arranged into four heptameric rings stacked in the order of α7β7β7α7 (2.Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. 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The binding of ubiquitinated substrates to proteasomes is facilitated through intrinsic ubiquitin receptors Rpn10, Rpn13, and Rpn1 of the base (7.Husnjak K. Elsasser S. Zhang N. Chen X. Randles L. Shi Y. Hofmann K. Walters K.J. Finley D. Dikic I. Proteasome subunit Rpn13 is a novel ubiquitin receptor.Nature. 2008; 453: 481-488Crossref PubMed Scopus (490) Google Scholar, 8.Chen X. Lee B.H. Finley D. Walters K.J. Structure of proteasome ubiquitin receptor hRpn13 and its activation by the scaffolding protein hRpn2.Mol. Cell. 2010; 38: 404-415Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 9.Elsasser S. Chandler-Militello D. Muller B. Hanna J. Finley D. Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome.J. Biol. Chem. 2004; 279: 26817-26822Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 10.Shi Y. Chen X. Elsasser S. Stocks B.B. Tian G. Lee B.H. Zhang N. de Poot S.A. Tuebing F. Sun S. Vannoy J. Tarasov S.G. Engen J.R. 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It has been an extremely challenging task to resolve the high-resolution structure of the 26S proteasome holocomplex because of compositional and conformational heterogeneity of the RPs. Recently, a series of Cryo-EM studies combined with X-ray crystallography and other biochemical experiments have revealed the molecular architectures of the yeast (4.Beck F. Unverdorben P. Bohn S. Schweitzer A. Pfeifer G. Sakata E. Nickell S. Plitzko J.M. Villa E. Baumeister W. Forster F. Near-atomic resolution structural model of the yeast 26S proteasome.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 14870-14875Crossref PubMed Scopus (216) Google Scholar, 5.Lander G.C. Estrin E. Matyskiela M.E. Bashore C. Nogales E. Martin A. Complete subunit architecture of the proteasome regulatory particle.Nature. 2012; 482: 186-191Crossref PubMed Scopus (475) Google Scholar, 6.Lasker K. Forster F. Bohn S. Walzthoeni T. Villa E. Unverdorben P. Beck F. Aebersold R. Sali A. Baumeister W. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 1380-1387Crossref PubMed Scopus (389) Google Scholar) and human 26S proteasomes (17.da Fonseca P.C. He J. Morris E.P. Molecular model of the human 26S proteasome.Mol Cell. 2012; 46: 54-66Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Most of the studies actually focused on the yeast proteasomes, whereas reports on the human 26S proteasome have been sparse. Only very recently, two high-resolution Cryo-EM structures (3.9 and 3.5 Å) of the human 26S proteasome were reported (18.Schweitzer A. Aufderheide A. Rudack T. Beck F. Pfeifer G. Plitzko J.M. Sakata E. Schulten K. Forster F. Baumeister W. Structure of the human 26S proteasome at a resolution of 3.9 A.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 7816-7821Crossref PubMed Scopus (145) Google Scholar, 19.Huang X. Luan B. Wu J. Shi Y. An atomic structure of the human 26S proteasome.Nat Struct Mol Biol. 2016; 23: 778-785Crossref PubMed Scopus (147) Google Scholar), indicating that the overall architecture of the 26S holocomplex is highly conserved from yeast to human. The six Rpt subunits of the 19S RP form a hexameric ring to associate with the cylinder ends of the 20S CP, and are surrounded by a shell of Rpn subunits (4.Beck F. Unverdorben P. Bohn S. Schweitzer A. Pfeifer G. Sakata E. Nickell S. Plitzko J.M. Villa E. Baumeister W. Forster F. Near-atomic resolution structural model of the yeast 26S proteasome.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 14870-14875Crossref PubMed Scopus (216) Google Scholar, 5.Lander G.C. Estrin E. Matyskiela M.E. Bashore C. Nogales E. Martin A. Complete subunit architecture of the proteasome regulatory particle.Nature. 2012; 482: 186-191Crossref PubMed Scopus (475) Google Scholar, 6.Lasker K. Forster F. Bohn S. Walzthoeni T. Villa E. Unverdorben P. Beck F. Aebersold R. Sali A. 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Molecular model of the human 26S proteasome.Mol Cell. 2012; 46: 54-66Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Subsequent studies revealed that limited number of particles and overestimated resolution led to the incorrect assignment of these subunits (4.Beck F. Unverdorben P. Bohn S. Schweitzer A. Pfeifer G. Sakata E. Nickell S. Plitzko J.M. Villa E. Baumeister W. Forster F. Near-atomic resolution structural model of the yeast 26S proteasome.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 14870-14875Crossref PubMed Scopus (216) Google Scholar, 5.Lander G.C. Estrin E. Matyskiela M.E. Bashore C. Nogales E. Martin A. Complete subunit architecture of the proteasome regulatory particle.Nature. 2012; 482: 186-191Crossref PubMed Scopus (475) Google Scholar), and that the subunit arrangement in the human proteasome is indeed identical to that in yeast (18.Schweitzer A. Aufderheide A. Rudack T. Beck F. Pfeifer G. Plitzko J.M. Sakata E. Schulten K. Forster F. Baumeister W. Structure of the human 26S proteasome at a resolution of 3.9 A.Proc. Natl. Acad. Sci. U.S.A. 2016; 113: 7816-7821Crossref PubMed Scopus (145) Google Scholar). Because of its structural dynamics, the proteasome exhibits a number of three-dimensional arrangements. Cryo-EM studies conducted in the presence of either ATPγS or ubiquitinated model substrates, along with a deep classification of a very large data set led us to identify coexisting conformational states and to define the conformational landscape of the 26S proteasome (20.Matyskiela M.E. Lander G.C. Martin A. Conformational switching of the 26S proteasome enables substrate degradation.Nat Struct Mol Biol. 2013; 20: 781-788Crossref PubMed Scopus (187) Google Scholar, 21.Forster F. Unverdorben P. Sledz P. Baumeister W. Unveiling the long-held secrets of the 26S proteasome.Structure. 2013; 21: 1551-1562Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22.Unverdorben P. Beck F. Sledz P. Schweitzer A. Pfeifer G. 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Despite advantages of XL-MS technologies, inherent challenges remain regarding unambiguous identification of cross-linked peptides because of complex fragmentation profiles of cross-linked peptides when conventional (i.e. noncleavable) cross-linkers are used. Each cross-linked peptide contains two covalently linked peptides, whose sequences have to be determined based on convoluted MS/MS spectra containing the fragments from the two linked sequences. In addition, the two linked peptides often yield inequitable numbers of sequence ions, thus preventing accurate identification of both peptides. Moreover, specialized database searching tools are required to properly determine cross-linked peptide sequences. Although new developments in bioinformatics tools have proven effective in identifying noncleavable cross-linked peptides (35.Walzthoeni T. Claassen M. Leitner A. Herzog F. Bohn S. Forster F. Beck M. Aebersold R. 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To this end, we have previously developed a suite of new MS-cleavable cross-linkers containing sulfoxide(s) groups within their spacer regions (e.g. disuccinimidyl sulfoxide (DSSO)) (34.Kaake R.M. Wang X. Burke A. Yu C. Kandur W. Yang Y. Novtisky E.J. Second T. Duan J. Kao A. Guan S. Vellucci D. Rychnovsky S.D. Huang L. A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.Mol. Cell. Proteomics. 2014; 13: 3533-3543Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 40.Kao A. Chiu C.L. Vellucci D. Yang Y. Patel V.R. Guan S. Randall A. Baldi P. Rychnovsky S.D. Huang L. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF Google Scholar, 41.Yu C. Kandur W. Kao A. Rychnovsky S. Huang L. 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Such fragmentation has proven robust and predictable, occurring independently of cross-linking types, peptide charges, and sequences, thus enabling simplified and accurate identification of sulfoxide-containing cross-linked peptides by MSn analysis and conventional database searching tools. DSSO is one of the amine-reactive sulfoxide-containing MS-cleavable cross-linkers that has been successfully applied for in vitro studies of purified protein complexes (27.Kao A. Randall A. Yang Y. Patel V.R. Kandur W. Guan S. Rychnovsky S.D. Baldi P. Huang L. Mapping the structural topology of the yeast 19S proteasomal regulatory particle using chemical cross-linking and probabilistic modeling.Mol. Cell. Proteomics. 2012; 11: 1566-1577Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 40.Kao A. Chiu C.L. Vellucci D. Yang Y. Patel V.R. Guan S. Randall A. Baldi P. Rychnovsky S.D. Huang L. 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