Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France.
Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):12011-6. doi: 10.1073/pnas.1204129109. Epub 2012 Jul 9.
Microtubules are cytoskeleton filaments consisting of αβ-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the β-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.
微管是由αβ-微管蛋白异二聚体组成的细胞骨架丝。它们在生长和收缩的相之间切换。这种被称为动态不稳定性的特性的潜在机制尚未完全了解。在这里，我们鉴定了一种设计的锚蛋白重复蛋白 (DARPin)，它以独特的方式干扰微管组装。其与 GTP-微管蛋白复合物的 X 射线结构表明，它结合到微管 (+) 末端暴露的β-微管蛋白表面。结构的细节提供了对 GTP 在微管聚合中的作用以及微管末端处微管蛋白构象状态的深入了解。它们特别表明，GTP 促进了伴随微管组装的微管蛋白结构转换，但不会在未聚合的微管蛋白中引发该转换。全内反射荧光显微镜显示，DARPin 通过选择性的末端封闭机制特异性地阻止微管 (+) 末端的生长，最终有利于从该末端进行微管解聚。DARPins 有望成为用于剖析微管动态特性的可设计工具，对微管的两个不同末端具有选择性。
