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破坏相互作用基序会增强设计的锚蛋白重复蛋白与微管蛋白的结合。

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

机构信息

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France.

Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.

出版信息

Sci Rep. 2016 Jul 6;6:28922. doi: 10.1038/srep28922.

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.

摘要

通过随机诱变和选择进行亲和力成熟是一种将结合分子变得更适合于生物医学研究、诊断和治疗应用的成熟技术。在这里,我们发现了一种意想不到的新型机制,即在体外进化微管蛋白特异性设计的锚蛋白重复蛋白(DARPin)时会增加亲和力。结构分析表明,在原代 DARPin 中,C 端封端重复序列(C-cap)发生 25°旋转,以避免在结合时与微管蛋白发生冲突。此外,C-cap 似乎参与与微管蛋白的静电排斥。生化和结构特征表明,进化突变体通过 C-cap 的去稳定化获得了亲和力的提高,这减轻了 DARPin 在结合微管蛋白时构象变化的需要,并消除了复合物中的不利相互作用。因此,这种特定的有序到无序的转变导致与微管蛋白形成了一个具有亚纳摩尔平衡解离常数的 100 倍更紧密的复合物,这与结合表面减少 30%显著相关。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7c91/4933879/ad2462336f5e/srep28922-f1.jpg

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