False discovery rate estimation for cross-linked peptides identified by mass spectrometry

Walzthoeni, Thomas; Claassen, Manfred; Leitner, Alexander; Herzog, Franz; Bohn, Stefan; Förster, Friedrich; Beck, Martin; Aebersold, Ruedi

doi:10.1038/nmeth.2103

Cited by 279 publications

(383 citation statements)

References 18 publications

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“…Although most of the detected crosslinks involve residues within unstructured domains, one crosslink occurs between lysines of Ubp6 and Rpt1 that are covered by the available atomic models (PDB ID codes 1VJV and 4CR3, respectively). The distance of the respective Cα-atoms is ∼28 Å for the best fit, which is below the cutoff of 30 Å previously applied for intersubunit crosslinks of the 26S proteasome (29); the distances are much larger for the majority of alternative solutions (Fig. 4).…”

Section: Analysis Of Conformational Ensembles and A Correlation Of Stmentioning

confidence: 61%

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Aufderheide

Beck

Stengel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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131

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In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediateenergy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.conformational switching | proteolysis | proteostasis | quality control D egradation of proteins that are misfolded, damaged, or no longer needed is an essential element of cellular homeostasis. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is the major pathway for regulated protein degradation (1). Proteins that are processed by the UPS are marked for destruction by polyubiquitin chains, which are recognized as a degradation signal by the 26S proteasome.The 26S proteasome consists of the core particle (CP), which degrades substrates into short peptides, and one or two 19S regulatory particles (RP), which associate with the ends of the cylinder-shaped CP to recruit substrates and prepare them for degradation (2, 3). Although the structure of the CP has been known for more than two decades (4, 5), the molecular architecture of the RP was unraveled by cryo-electron microscope (EM)-based approaches only recently (6-9). It comprises six RP triple A (AAA) ATPases (Rpt), 1-6, and 13 RP non-ATPases (Rpn), 1-3, 5-13, and 15. Similar to AAA-ATPases in prokaryotic ATP-dependent proteases, the Rpts form a hexameric ring that binds to the ends of the CP and is responsible for substrate unfolding and translocation into the CP. Unlike their prokaryotic counterparts, the Rpts are surrounded by non-ATPases. Apart from Rpn1, all Rpns form a cohesive struct...

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Section: Analysis Of Conformational Ensembles and A Correlation Of Stmentioning

confidence: 61%

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Aufderheide

Beck

Stengel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

131

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“…To assign the fragment ion spectra, raw files were converted to centroid mzXML using the Mass Matrix file converter tool and then searched using xQuest (Leitner et al , 2014) against a fasta database containing the sequences of the cross‐linked proteins. Posterior probabilities were calculated using xProphet (Walzthoeni et al , 2012). …”

Section: Methodsmentioning

confidence: 99%

Structural insights into transcription initiation by yeast RNA polymerase I

et al. 2017

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In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre‐rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi‐subunit pre‐initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo‐EM structure of the 18‐subunit yeast Pol I PIC bound to a transcription scaffold. The cryo‐EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB‐related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

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“…0.9 mg purified ITC (1.2 mg ml À 1 ) was incubated with an eightfold molar excess of DNA-RNA scaffold and crosslinked with 0.6 mM isotope-labeled disuccimidyl suberate (DSS-d0/d12, Creative Molecules Inc.) as described 13 . Crosslinked protein was digested, and the crosslinked peptides were enriched, analysed by liquid chromatography coupled to tandem mass spectrometer (Orbitrap Elite), and spectra were searched by the xQuest software 31,32 . The resulting cross-link identifications were manually validated and the local false discovery rates for each individual cross-link were estimated as described (Supplementary Table 1).…”

Section: Methodsmentioning

confidence: 99%

Conserved architecture of the core RNA polymerase II initiation complex

et al. 2014

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During transcription initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core initiation complex that recruits additional factors. The core complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II initiation complex during transcription initiation. The yeast complex resembles the human initiation complex and reveals that the TFIIF Tfg2 winged helix domain swings over promoter DNA. An 'arm' and a 'charged helix' in TFIIF function in transcription start site selection and initial RNA synthesis, respectively, and apparently extend into the active centre cleft. Our model provides the basis for further structure-function analysis of the entire transcription initiation complex.

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False discovery rate estimation for cross-linked peptides identified by mass spectrometry

Cited by 279 publications

References 18 publications

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Structural insights into transcription initiation by yeast RNA polymerase I

Conserved architecture of the core RNA polymerase II initiation complex

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