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Science 337 (6100): 1348-1352

Copyright © 2012 by the American Association for the Advancement of Science

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

Franz Herzog1,*, Abdullah Kahraman1,*, Daniel Boehringer2,*, Raymond Mak1, Andreas Bracher4, Thomas Walzthoeni1, Alexander Leitner1, Martin Beck3, Franz-Ulrich Hartl4, Nenad Ban2, Lars Malmström1, and Ruedi Aebersold1,{dagger}

1 Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, Wolfgang-Pauli Strasse 16, 8093 Zurich, Switzerland.
2 Department of Biology, Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zürich, Schafmattstrasse 20, 8093 Zurich, Switzerland.
3 European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany.
4 Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

Figure 1
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Fig. 1. Schematic outline of the XL-MS workflow established for the identification of chemically cross-linked lysines on affinity-purified human PP2A complexes (16). LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LIT, linear ion trap; TPP, trans-proteomic pipeline.


Figure 2
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Fig. 2. Identification of interprotein distance restraints on PP2A protein complexes by XL-MS. (A) PP2A protein interaction network based on reciprocal pull-down assays. (B) PP2A network topology represented by interprotein cross-links. [(A) and (B)] Network is split according to PP2A regulatory subunits. B' (2A5A, 2A5B, 2A5D, 2A5E, and 2A5G) subunits are shown in the left panel and B (2ABA, 2ABD, and 2ABG), B" (P2R3C) and B"' (STRN, STRN3, and STRN4) in the right panel. (C) Histogram of the Euclidean Cα-Cα distances between cross-linked lysines determined on x-ray structures and comparative models of the PP2A network. Threshold, estimated maximum Cα-Cα distance spanned by the cross-linking reagent.


Figure 3
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Fig. 3. Topology of PP2A-associated protein complexes by applying cross-links to guide computational modeling and docking. (A to C) Identification of the IGBP1-PP2AA binding interface using distance restraints in protein-protein docking experiments (13). (A) Docking models of IGBP1 and PP2AA by superimposing 237 IGBP1 cluster representatives (green) on a PP2AA/2AAA/2A5G complex (PDB entry 3FGA). (B) Four cluster representatives of IGBP1-PP2AA docking models with the shortest average distance for interprotein cross-links. (C) Frequency of amino acid residues mapped to the interface in IGBP1-PP2AA docking models. (D) Pull-down (PD) binding assay. IGBP1 wild-type and mutant proteins were affinity-purified from human cell extracts (xt), and binding of PP2A catalytic (C) subunits was analyzed by immunoblotting. (E) Schematic subunit map of the STRIPAK complex based on interprotein cross-links. (F) Interprotein cross-links detected on a complex of SGOL1 and SET suggest an antiparallel arrangement. (G) Hydrophobic residues (green spheres) essential for SGOL1 homodimerization (PDB entry 3FGA). (H) Scheme of SGOL1 in complex with SET based on cross-links. Green spheres, hydrophobic residues.


Figure 4
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Fig. 4. Architecture of the TRiC chaperonin bound to its substrate 2ABG. (A) The subunit register of the two stacked TRiC rings was indicated by 19 inter-ring cross-links. (B) Eleven lysine residues (black spheres) within six TRiC subunits were cross-linked to 2ABG. (C) Schematic model of TRiC in complex with its substrate 2ABG based on cross-links. (D) Cryo-EM 3D-reconstruction of the 2ABG-TRiC complex. The comparative model of human TRiC (blue) was fitted into the EM 3D structure of TRiC (yellow). A globular density (red mesh representation) was located at the 2ABG position inferred from cross-links.


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