title: Small-Molecule-Induced and Cooperative Enzyme Assembly on a 14-3-3 Scaffold.
abstract: Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems.
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Anniek
authors.initials: A
authors.lastname: den Hamer
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Lenne J M
authors.initials: LJ
authors.lastname: Lemmens
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Minke A D
authors.initials: MA
authors.lastname: Nijenhuis
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Christian
authors.identifier: 0000-0001-7315-0315
authors.initials: C
authors.lastname: Ottmann
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Maarten
authors.identifier: 0000-0001-9484-3882
authors.initials: M
authors.lastname: Merkx
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Tom F A
authors.identifier: 0000-0002-9338-284X
authors.initials: TF
authors.lastname: de Greef
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering and, Institute of Complex Molecular Systems, Eindhoven University of Technology, Den Dolech 2, 5612, AZ Eindhoven, Netherlands.
authors.forename: Luc
authors.identifier: 0000-0001-5675-511X
authors.initials: L
authors.lastname: Brunsveld
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D048948:14-3-3 Proteins; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D053148:Caspase 3; D053453:Caspase 9
keywords: 14-3-3 proteins; combinatorial inhibition; cooperative effects; dimerization; protein engineering; protein scaffolds