Structural Probing of a Protein Phosphatase 2A Network by Chemical Cross-Linking and Mass Spectrometry

Herzog, Franz; Kahraman, Abdullah; Boehringer, Daniel; Mak, Raymond; Bracher, A.; Walzthoeni, Thomas; Leitner, Alexander; Beck, Martin; Hartl, F. Ulrich; Ban, Nenad; Malmström, Lars; Aebersold, Ruedi

doi:10.1126/science.1221483

Cited by 362 publications

(426 citation statements)

References 85 publications

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“…The BzF cross-linking established contacts between the Gβ N-terminal α-helix and CCTγ, but gave no information about the orientation and interactions of the β-propeller region. To address these issues, we used XL-MS, a method that has been used to characterize CCT/substrate complexes (14). The Gβ-CCT complex was treated with disuccinimidyl suberate (DSS), which cross-links adjacent lysine residues.…”

Section: Resultsmentioning

confidence: 99%

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Plimpton

Cuéllar

Lai

et al. 2015

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

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G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.G-protein | chaperonin | phosducin-like protein | electron cryo-microscopy | cross-linking

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Section: Resultsmentioning

confidence: 99%

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Plimpton

Cuéllar

Lai

et al. 2015

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

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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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“…Of the 138 lysines in the Ndc80 complex, 128 were identified in mono-links (93%; Figure S2B), suggesting that we have nearly saturated available reactive sites with the cross-linking reagent. Structural information can be determined from the cross-linked peptides, which represent pairs of primary amine groups whose backbone a-carbons are within $30 Å of one another in the three-dimensional structure of the complex (Herzog et al 2012). In total, our approach revealed 277 unique crosslinks and 85 unique loop-links within the Ndc80 complex with $95%…”

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confidence: 99%

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

et al. 2014

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Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore-microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore-microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis. KINETOCHORES mediate the linkage between chromosomes and spindle microtubules during mitosis. This attachment is highly regulated to promote the fidelity of chromosome segregation. At metaphase, replicated chromosomes are attached to microtubules emanating from opposite poles, in a "bioriented" alignment. This ensures that each daughter cell receives a full complement of genetic material after chromosome segregation. Errors can occur in the form of "syntelic" attachments, when both sister kinetochores attach to microtubules emanating from the same pole. These erroneous kinetochore-microtubule attachments are detected and detached by the conserved Aurora B/Ipl1 kinase Tanaka et al. 2002;Pinsky et al. 2006). In the current prevailing model, Aurora B/Ipl1 corrects syntelic attachments by destabilizing linkages that are under low levels of tension (Nicklas and Koch 1969;Tanaka et al. 2002;Liu et al. 2009;Cane et al. 2013). The Aurora B/Ipl1 error detection system is coupled to the spindle checkpoint, a separate surveillance system that delays anaphase until all kinetochores are attached to spindle microtubules (reviewed in Hauf 2013). Together, these systems ensure that the physical separation of replicated chromatids does not occur until all kinetochore-microtubule attachments are bioriented.The attachment of spindle microtubules to kinetochores requires the conserved Ndc80 complex ( Figure 1A) rod-sh...

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Structural Probing of a Protein Phosphatase 2A Network by Chemical Cross-Linking and Mass Spectrometry

Cited by 362 publications

References 85 publications

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Conserved architecture of the core RNA polymerase II initiation complex

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

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