5EYL

TUBULIN-BINDING DARPIN

5EYL の概要

• エントリーDOI: 10.2210/pdb5eyl/pdb
• 分子名称: DESIGNED ANKYRIN REPEAT PROTEIN (DARPIN), PHOSPHATE ION, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: darpin, microtubule, tubulin, protein binding
• 由来する生物種: synthetic construct
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 36560.16

構造登録者

Ahmad, S.,Kossow, M.,Gigant, B. (登録日: 2015-11-25, 公開日: 2016-07-20, 最終更新日: 2024-01-10)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.41 Å)