5EYP

TUBULIN-DARPIN COMPLEX

Summary for 5EYP

• Entry DOI: 10.2210/pdb5eyp/pdb
• Related: 5EYL
• Descriptor: Tubulin alpha chain, Tubulin beta chain, DESIGNED ANKYRIN REPEAT PROTEIN (DARPIN), ... (10 entities in total)
• Functional Keywords: darpin, microtubule, tubulin, protein binding, cell cycle
• Biological source: synthetic construct; More
• Cellular location: Cytoplasm, cytoskeleton : D0VWZ0 D0VWY9
• Total number of polymer chains: 3
• Total formula weight: 120574.66

Authors

Ahmad, S.,Knossow, M.,Gigant, B. (deposition date: 2015-11-25, release date: 2016-07-20, Last modification date: 2024-01-10)

Primary citation

Ahmad, S.,Pecqueur, L.,Dreier, B.,Hamdane, D.,Aumont-Nicaise, M.,Pluckthun, A.,Knossow, M.,Gigant, B.
Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.
Sci Rep, 6:28922-28922, 2016

PubMed 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.

PubMed: 27380724
DOI: 10.1038/srep28922

Experimental method

X-RAY DIFFRACTION (1.9 Å)