Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex.
Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 CNRS/Université de Strasbourg - "Chimie de la Matière Complexe", 1 Rue Blaise Pascal, 67008, Strasbourg, France.
Nha-Thi
NT
Nguyen-Huynh
Integrated Structural Biology Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404, Illkirch, France.
Grigory
G
Sharov
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 CNRS/Université de Strasbourg - "Chimie de la Matière Complexe", 1 Rue Blaise Pascal, 67008, Strasbourg, France.
Clément
C
Potel
Integrated Structural Biology Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404, Illkirch, France.
Pélagie
P
Fichter
European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France.
Simon
S
Trowitzsch
European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France.
Imre
I
Berger
Integrated Structural Biology Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404, Illkirch, France.
Valérie
V
Lamour
Integrated Structural Biology Department, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404, Illkirch, France.
Patrick
P
Schultz
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 CNRS/Université de Strasbourg - "Chimie de la Matière Complexe", 1 Rue Blaise Pascal, 67008, Strasbourg, France.
Noëlle
N
Potier
Laboratoire de Spectrométrie de Masse des Interactions et des Systèmes (LSMIS) UMR 7140 CNRS/Université de Strasbourg - "Chimie de la Matière Complexe", 1 Rue Blaise Pascal, 67008, Strasbourg, France.
Emmanuelle
E
Leize-Wagner
D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013388:Succinimides / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D050880:p300-CBP Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D048868:Adaptor Proteins, Signal Transducing; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D013388:Succinimides; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C534292:TADA2B protein, human; C444103:TADA3 protein, human; C035760:bis(sulfosuccinimidyl)suberate; C561317:Sgf29 protein, human
SAGA HAT subcomplex; cross-linking mass spectrometry; protein interaction; protein-; proteomics