title: Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.
abstract: Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
authors.forename: Shoeb
authors.initials: S
authors.lastname: Ahmad
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Pecqueur
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
authors.forename: Birgit
authors.initials: B
authors.lastname: Dreier
authors.affiliation: Laboratoire de Chimie des Processus Biologiques, CNRS-UMR 8229, Collège De France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France.
authors.forename: Djemel
authors.initials: D
authors.lastname: Hamdane
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Aumont-Nicaise
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
authors.forename: Marcel
authors.initials: M
authors.lastname: Knossow
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Gigant
mesh_terms: D020816:Amino Acid Motifs; D017089:Ankyrin Repeat; D017487:Ankyrins / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D012270:Ribosomes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014404:Tubulin / Q000737:chemistry*
chemical_list: D017487:Ankyrins; D029968:Escherichia coli Proteins; D014404:Tubulin