Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics

Leitner, Alexander; Walzthoeni, Thomas; Kahraman, Abdullah; Herzog, Franz; Rinner, Oliver; Beck, Martin; Aebersold, Ruedi

doi:10.1074/mcp.r000001-mcp201

Cited by 423 publications

(462 citation statements)

References 82 publications

(112 reference statements)

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“…On a specific full-particle model (arrangement), any cross-link can be assigned a distance between the C α atoms of the cross-linked lysine residues. Previous studies of crosslinked proteins with known crystal structures have established that this distance is less than 28Ǻ for most cross-links with few instances being as high as 33Ǻ because of local protein flexibility (21,25,31). These numbers are in complete accord with the cross-linker length plus twice the length of the lysine side chain.…”

Section: Resultssupporting

confidence: 62%

“…An alternative biochemical approach that analyzed the content of spontaneously occurring TRiC fragments (19) suggested a possible ring arrangement that has not been reaffirmed so far. Furthermore, all previous suggestions for the particle arrangement are completely incompatible with our recent inter-subunit interface analysis that is based on evolutionary conservation (20).An emerging structure determination technique that has the potential to conclusively determine the subunit order in TRiC is cross-linking coupled with mass spectrometry (MS) (21,22). In this technique, the complex is incubated under native conditions with a cross-linker that is capable of forming specific covalent bonds with side chains on the surface of the complex.…”

mentioning

confidence: 80%

“…An emerging structure determination technique that has the potential to conclusively determine the subunit order in TRiC is cross-linking coupled with mass spectrometry (MS) (21,22). In this technique, the complex is incubated under native conditions with a cross-linker that is capable of forming specific covalent bonds with side chains on the surface of the complex.…”

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confidence: 99%

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Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Kalisman

Adams

Levitt

2012

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

163

178

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The TRiC/CCTchaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent crosslinking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (¼8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.group II chaperonins | protein folding | violation distances I n eukaryotes and archaea the folding of nascent and mis-folded polypeptide chains is assisted by a group II chaperonin system known as the thermosome in archea and TRiC (or CCT) in eukaryotes (1). These large protein complexes consist of two stacked octameric rings (2) The open state of the complex binds the polypeptide substrate (4, 5), whereupon closing the substrate is sequestered into a large interior cavity where folding can occur. TRiC has been implicated in the folding pathways of many cytosolic proteins (6), most notably actin and tubulin (7,8).Many archaeal species have just one thermosome gene and a simple homo-oligomeric architecture (9). Other archaeal species have at most three different types of subunits, and there is evidence that the multiple genes are redundant (10). In stark contrast, the eukaryotic TRiC consists of eight different subunit types (CCT1 to CCT8), all of which are essential. The subunit specialization occurred very early in eukaryote evolution (11) and is conserved to such an extent that the sequence identity between mammalian and yeast subunits of the same type is nearly 60%, whereas the sequence identity between the different subunit types in the same organism is only 30%. The diversity of eight distinct subun...

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Section: Resultssupporting

confidence: 62%

mentioning

confidence: 80%

mentioning

confidence: 99%

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Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Kalisman

Adams

Levitt

2012

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

163

178

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“…We have used crosslinking mass spectrometry, i.e., the mass spectrometric detection of crosslinked peptides, to investigate crosslinks between proteins. This approach (also called CXMS) is currently maturing into a useful approach to characterize proteinprotein interactions, even though some technical obstacles still prevent its exploitation to its full potential (Leitner et al 2010;Singh et al 2010;Sinz 2006). Our previously developed workflow, using lysine-specific crosslinking, offline nanoliquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight (LC MALDI-TOF/TOF) mass spectrometry and an in-house developed program FINDX, was shown to allow the identification of true crosslinks that are all within crosslinking distance (<20 Å), mapping well into the structure of a protein oligomer (Lambert et al 2011b;Söderberg et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

Lambert

Rutsdottir

Hussein

et al. 2013

Cell Stress and Chaperones

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Small heat-shock protein chaperones are important players in the protein quality control system of the cell, because they can immediately respond to partially unfolded proteins, thereby protecting the cell from harmful aggregates. The small heat-shock proteins can form large polydisperse oligomers that are exceptionally dynamic, which is implicated in their function of protecting substrate proteins from aggregation. Yet the mechanism of substrate recognition remains poorly understood, and little is known about what parts of the small heat-shock proteins interact with substrates and what parts of a partially unfolded substrate protein interact with the small heat-shock proteins. The transient nature of the interactions that prevent substrate aggregation rationalize probing this interaction by crosslinking mass spectrometry. Here, we used a workflow with lysine-specific crosslinking and offline nano-liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to explore the interaction between the plant small heat-shock protein Hsp21 and a thermosensitive model substrate protein, malate dehydrogenase. The identified crosslinks point at an interaction between the disordered N-terminal region of Hsp21 and the C-terminal presumably unfolding part of the substrate protein.

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“…To obtain information on protein-protein proximities at the peptide level, we chemically cross-linked the purified S. pombe 26S proteasomes using the lysine reactive cross-linker disuccinimidyl suberate (DSS), subjected the trypsin-digested samples to liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identified the cross-linked peptides from MS/ MS spectra at a false-discovery rate (FDR) of less than 5% (Materials and Methods) (30). Thirty-two cross-links were obtained within the AAA-ATPase hexamer and four between the CP and the AAA-ATPases, involving nine and three different subunit pairs, respectively ( Fig.…”

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confidence: 99%

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

Lasker

Förster

Bohn

et al. 2012

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

Self Cite

428

513

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The 26S proteasome is at the executive end of the ubiquitinproteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAAATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.coiled coils | mass spectrometry | proteasome-COP9-eIF3 domain | proteasome-cyclosome repeats I n eukaryotes, the ubiquitin-proteasome pathway (UPP) is essential for proteostasis: Misfolded proteins or otherwise defective proteins as well as short-lived regulatory proteins are eliminated by degradation (1). The UPP regulates many fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (for review see ref.2). The 26S proteasome is the most downstream element of the UPP, executing the degradation of polyubiquitylated substrates (3-5). It consists of the barrelshaped core particle (CP; approximately 700 kDa), which sequesters the proteolytically active site in its central cavity, and the regulatory particle (RP; approximately 900 kDa), which is attached at either one or both ends of the CP and prepares substrates for degradation (6).The RP consists of 19 different canonical subunits, including six regulatory particle AAA-ATPase subunits (Rpt1-6) and 13 regulatory particle non-ATPase subunits (Rpn1-3, Rpn5-13, and Rpn15). The integral ubiquitin (Ub) receptors Rpn10 and Rpn13 recognize polyubiquitylated substrates (7-9). Alternatively, polyubiquitylated substrates can be recruited by shuttling Ub-receptors (Dsk2, Rad23, Ddi2), which bind to substrates with their Ub-associated domain, and to Rpn1, Rpn10, or Rpn13 at their Ub-like domain (5). The metalloprotease Rpn11 deubiquitylates substrates prior to their degradation (10, 11). The functions of the other Rpn subunits remain to be established. The AAA-ATPases form a hexameric ring that unfolds substrates, opens the gate to the CP, and eventually translocates the substrates to the CP.Electron microscopy (EM) (12) and X-...

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Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics

Cited by 423 publications

References 82 publications

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

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