Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin - Laboratoire de Chimie des Processus Biologiques Accéder directement au contenu
Article Dans Une Revue Scientific Reports Année : 2016

Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin

Résumé

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.
Fichier principal
Vignette du fichier
srep28922.pdf (5.03 Mo) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte

Dates et versions

hal-01356073 , version 1 (25-01-2024)

Licence

Paternité

Identifiants

Citer

Shoeb Ahmad, Ludovic Pecqueur, Birgit Dreier, Djemel Hamdane, Magali Aumont-Nicaise, et al.. Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin. Scientific Reports, 2016, 6, pp.28922. ⟨10.1038/srep28922⟩. ⟨hal-01356073⟩
183 Consultations
5 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More