pFind Studio: a computational solution for mass spectrometry-based proteomics
2020
Cell2020. Mashtalir, N et al.
Dana Farber Canc Inst, Dept Pediat Oncol, Boston, MA 02115 USA.
ABSTRACT:Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal alpha-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.
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Nature2020. Haibo Wang et al.
Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Gttingen, Germany
ABSTRACT:Genetranscriptionby RNA polymerase II is regulated by activator proteins that recruitthecoactivatorcomplexesSAGA(Spt-Ada-Gcn5-acetyltransferase)(1,2) andtranscriptionfactor IID (TFIID)(2-4).SAGAis required for all regulatedtranscription(5) and is conserved among eukaryotes(6).SAGAcontains four modules(7-9):theactivator-binding Tra1 module,thecore module,thehistone acetyltransferase (HAT) module andthehistone deubiquitination (DUB) module. Previous studies provided partial structures(10-14), butthestructureofthecentral core module is unknown. Here we presentthecryo-electron microscopystructureofSAGAfromtheyeast Saccharomyces cerevisiae and resolvethecore module at 3.3 angstrom resolution.Thecore module consistsofsubunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold.Theoctamer-like fold comprisestheheterodimers Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 andtheadjacent subunit Spt8 interact withtheTATA box-binding protein (TBP)(2,7,15-17).Theoctamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 andtheTaf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections totheTra1 module, whereas Sgf73 tetherstheDUB module. Bindingofa nucleosome toSAGAdisplacestheHAT and DUB modules fromthecore-module surface, allowingtheDUB module to bind one faceofan ubiquitinated nucleosome.
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Nature Structural & Molecular Biology2020. Vos, SM et al.
Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT:Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four alpha-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation. Cryo-EM elucidation of a fully reconstituted Pol II-DSF-PAF1-SPT6 elongation complex defines the position of PAF1 subunit RTF1 and reveals contacts with the Pol II bridge helix that may allosterically stimulate transcription elongation.
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Nature2020. Wagner, FR et al.
Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT:The cryo-electron microscopy structure of the 16-subunit yeast SWI/SNF complex RSC in complex with a nucleosome substrate provides insights into the chromatin-remodelling function of this family of protein complexes. Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)(1,2). In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth1(4,5). RSC removes nucleosomes from promoter regions(6,7) and positions the specialized +1 and -1 nucleosomes that flank NDRs(8,9). Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements(8,10,11) that influence RSC functionality(12). The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity(5). The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer(13).
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Science (New York, N.Y.)Y.). 2020. Townsend, Cole et al.
Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Gttingen, Germany.
ABSTRACT:Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA. Copyright 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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Nature2020. Zhang, ZW et al.
MPI Biophys Chem, Dept Struct Dynam, Gottingen, Germany.
ABSTRACT:The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing'. Stable addition of U2 during early spliceosome formation requiresthe DEAD-box ATPase PRP5(2-7). Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)(8), but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers(9), contains a HEAT domain (SF3B1(HEAT)) with an open conformation in isolated SF3b(10), but a closed conformation in spliceosomes(11), which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure ofthe human 17S U2 snRNP at a core resolution of 4.1 angstrom and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1(HEAT) interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1(HEAT). Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.
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Nature Communications2020. Beveridge, R et al.
Vienna Bioctr VBC, Res Inst Mol Pathol IMP, Campus Vienna Bioctr 1, A-1030 Vienna, Austria.
ABSTRACT:Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%. We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.
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Nature chemical biology2020. Dao, HT et al.
Princeton Univ, Dept Chem, Princeton, NJ USA.
ABSTRACT:A photocrosslinking-based nucleosome profiling approach is used to identify a conserved basic motif in the ISWI remodeler SNF2h that anchors it to the acidic patch of nucleosome and enables nucleosome sliding activity. Recent studies have implicated the nucleosome acidic patch in the activity of ATP-dependent chromatin remodeling machines. We used a photocrosslinking-based nucleosome profiling technology (photoscanning) to identify a conserved basic motif within the catalytic subunit of ISWI remodelers, SNF2h, which engages this nucleosomal epitope. This region of SNF2h is essential for chromatin remodeling activity in a reconstituted biochemical system and in cells. Our studies suggest that the basic motif in SNF2h plays a critical role in anchoring the remodeler to the nucleosomal surface. We also examine the functional consequences of several cancer-associated histone mutations that map to the nucleosome acidic patch. Kinetic studies using physiologically relevant heterotypic nucleosomal substrates ('Janus' nucleosomes) indicate that these cancer-associated mutations can disrupt regularly spaced chromatin structure by inducing ISWI-mediated unidirectional nucleosome sliding. These results indicate a potential mechanistic link between oncogenic histones and alterations to the chromatin landscape.
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Nature communications2020. Shang, JS et al.
Scripps Res Inst, Dept Integrat Struct & Computat Biol, Jupiter, FL 33458 USA.
ABSTRACT:Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPAR gamma) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume. Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations-supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations. Structural studies of nuclear receptor transcription factors revealed that nearly all nuclear receptors share a conserved helix 12 dependent transcriptional activation mechanism. Here the authors present two crystal structures of peroxisome proliferator-activated receptor gamma (PPAR gamma) in an inverse agonist/corepressor-bound transcriptionally repressive conformation, where helix 12 is located within the orthosteric ligand-binding pocket instead, and discuss mechanistic implications.
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PNAS2020. Mintseris, J et al.
Harvard Med Sch, Dept Cell Biol, Boston, MA 02115 USA.
ABSTRACT:Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are selfconsistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique crosslinked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.
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