Rice Luke M, Montabana Elizabeth A, Agard David A
Department of Biochemistry and Biophysics and Howard Hughes Medical Institute, University of California, San Francisco, CA 94158-2517, USA.
Proc Natl Acad Sci U S A. 2008 Apr 8;105(14):5378-83. doi: 10.1073/pnas.0801155105. Epub 2008 Apr 3.
GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alphabeta-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alphabeta-tubulin. The allosteric model posits that unpolymerized alphabeta-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alphabeta-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alphabeta-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alphabeta-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alphabeta-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior.
细胞分裂需要GTP依赖的微管聚合动力学，并且在聚合亚基αβ-微管蛋白中伴随着结构域重排。两种相反的模型描述了GTP的作用及其与αβ-微管蛋白构象变化的关系。变构模型假定未聚合的αβ-微管蛋白在结合GTP后会采取更具聚合能力的构象。晶格模型假定构象变化仅在被招募到正在生长的晶格中时才会发生。已发表的数据支持晶格模型，但大多是间接的，因此变构模型占了上风。我们提出了两种独立的αβ-微管蛋白构象的溶液探针，与GDP结合的γ-微管蛋白的2.3埃晶体结构，以及动力学模拟以解释结构数据的功能后果。这些结果（结合我们之前的γ-微管蛋白：GTPγS结构）通过证明真核微管蛋白不会因GTP结合而发生主要结构域重排，以及αβ-微管蛋白的未聚合构象与聚合构象有显著差异，从而支持了晶格模型。因此，横向自组装的几何约束必须驱动αβ-微管蛋白的构象变化，而GTP在调节微管晶格内纵向接触强度方面起次要作用。αβ-微管蛋白的行为就像一个弯曲的弹簧，抵抗伸直，直到通过与正在生长的微管的GTP介导的相互作用被迫伸直。动力学模拟表明，对伸直的阻力通过特异性地破坏对横向相互作用强度特别敏感的早期组装中间体而阻碍微管起始。这些数据为动态微管行为的分子起源提供了新的见解。
