Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.
Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.
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DOI：
10.1073/pnas.2114994119
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发表时间：
2022-01-11
影响因子：
11.1
通讯作者：
Nogales E
中科院分区：
文献类型：
--
作者：
LaFrance BJ;Roostalu J;Henkin G;Greber BJ;Zhang R;Normanno D;McCollum CO;Surrey T;Nogales E
微管（MT）是在生长和收缩状态之间切换的非平衡聚合物。这种性质对于它们的功能至关重要，并且是MT中GTP水解的结果。MT的稳定GTP部分（GTP帽）的结构先前已从与不可水解的GTP类似物聚合的MT推断。在这里，我们报告的高分辨率结构的MT真正含有GTP，聚合突变，水解缺陷的微管蛋白。我们发现，GTP-MTs有一个“扩展的晶格”和“封闭的缝”，结构特征可能负责稳定GTP帽。这些结果提供了深入了解在不断增长的MT端的结构转变，进一步加深了我们的理解MT的可移植性。微管（MT）是αβ-微管蛋白异源二聚体的聚合物，其在生长和收缩阶段之间随机切换。这种动态不稳定性对MT功能至关重要。据信，在MT晶格内的GTP水解伴随着不稳定的构象变化，并且MT稳定性取决于在生长的MT末端的瞬时存在的GTP帽。在这里，我们使用冷冻电子显微镜和全内反射荧光显微镜的GTP水解缺陷的MT突变重组人微管蛋白组装调查的GTP结合MT晶格的结构。我们发现两个突变体（E254 A和E254 N）的GTP-MT晶格具有明显的可塑性，其中α-微管蛋白中催化活性的谷氨酸被非活性氨基酸取代。具有13个原丝的未修饰的E254 A和E254 N MT都具有扩展的晶格，但显示相反的原丝扭曲，使得这些晶格不同于野生型GDP-MT的紧凑晶格。EB家族的末端结合蛋白具有紧凑突变GTP晶格和稳定负扭转的能力，这表明它们也在野生型MT的GTP帽中促进这种转变，从而有助于MT结构的成熟。我们还发现，MT接缝似乎是稳定的突变体GTP-MT和不稳定的GDP-MT，支持的建议，接缝在MT稳定性中起着重要作用。总之，这些结构的催化非活性的MT添加机制洞察到GTP状态的MT，GTP和GDP结合的晶格的稳定性，和我们的整体理解MT动态不稳定性。
Microtubules (MTs) are nonequilibrium polymers that switch between states of growth and shrinkage. This property is critical for their function and is a consequence of GTP hydrolysis in the MT. The structure of the stable GTP part of the MT (the GTP cap) has previously been inferred from MTs polymerized with nonhydrolyzable GTP analogs. Here, we report high-resolution structures of MTs truly containing GTP, polymerized from mutated, hydrolysis-deficient tubulins. We find that GTP-MTs have an “expanded lattice” and a “closed seam,” structural characteristics possibly responsible for stabilizing the GTP cap. These results provide insight into the structural transitions at growing MT ends, furthering our understanding of the bistable nature of MTs. Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.
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DOI：
10.1107/s2059798318009324
发表时间：
2018-09-01
期刊：
Acta crystallographica. Section D, Structural biology
影响因子：
--
作者：
Afonine PV;Klaholz BP;Moriarty NW;Poon BK;Sobolev OV;Terwilliger TC;Adams PD;Urzhumtsev A
通讯作者：
Urzhumtsev A
影响因子：
5.6
作者：
Kellogg, Elizabeth H.;Hejab, Nisreen M. A.;Nogales, Eva
通讯作者：
Nogales, Eva
高分辨率微管结构揭示了 GTP 水解后 αβ-微管蛋白的结构转变。
影响因子：
64.5
作者：
Alushin GM;Lander GC;Kellogg EH;Zhang R;Baker D;Nogales E
通讯作者：
Nogales E
影响因子：
3.3
作者：
Chaaban S;Brouhard GJ
通讯作者：
Brouhard GJ
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH