Unveiling the catalytic mechanism of GTP hydrolysis in microtubules.
Unveiling the catalytic mechanism of GTP hydrolysis in microtubules.
复制标题
DOI：
10.1073/pnas.2305899120
复制
发表时间：
2023-07-04
影响因子：
11.1
通讯作者：
Voth, Gregory A.
中科院分区：
文献类型：
--
作者：
Beckett, Daniel;Voth, Gregory A.
微管是真核细胞骨架的大而动态的组成部分，能够随机地从聚合状态转换到解聚状态，反之亦然。解聚与鸟苷-5‘-三磷酸(GTP)的水解有关，在MT晶格中的GTP比在游离微管蛋白杂二聚体中快几个数量级。我们的结果从计算上确定了与自由杂二聚体相比，MT晶格中催化残基接触加速了GTP的水解，并证实了紧凑的MT晶格是水解所必需的，而更膨胀的晶格不能形成必要的接触从而水解GTP。微管是由αβ-微管蛋白杂二聚体组成的大分子细胞骨架聚合物，能够随机地从聚合态转化为解聚态，反之亦然。解聚与β-微管蛋白中的鸟苷三磷酸(GTP)的水解相耦合。与自由杂二聚体相比，MT晶格中的水解更有利，实验观察到的速率增加了500-700倍，相应地，动能垒降低了3.8-4.0kcal/mol。突变研究表明，α-微管蛋白残基α：E254和α：D251是完成MT晶格中较低杂二聚体的β-微管蛋白活性部位的催化残基。然而，GTP在游离杂二聚体中的水解机理尚不清楚。此外，关于GTP状态晶格相对于GDP状态是扩展的还是紧凑的，以及是否需要“紧凑的”GDP状态晶格来进行水解，一直存在争议。在这项工作中，我们进行了大量的量子力学/分子力学模拟，包括紧凑和扩展的中间二聚体络合物以及自由杂二聚体的过渡回火亚动力学自由能采样，以提供对GTP水解机理的清楚了解。α：E254是致密晶格中的催化残基，而在扩展晶格中，关键的盐桥相互作用的破坏使α：E254的催化活性降低。模拟结果表明，与自由异质二聚体相比，致密晶格的势垒降低了3.8±0.5kcal/mol，与实验动力学测量结果一致。此外，膨胀的晶格势垒比压实的高6.3±0.5kcal/mol，这表明GTP的水解是随晶格状态而变化的，并且在MT尖端的水解速度较慢。
Microtubules (MTs) are large and dynamic components of the eukaryotic cytoskeleton with the ability to stochastically convert from a polymerizing to a depolymerizing state and vice versa. Depolymerization is coupled to the hydrolysis of guanosine-5′-triphosphate (GTP), which is orders of magnitude faster in the MT lattice than in free tubulin heterodimers. Our results computationally ascertain the catalytic residue contacts in the MT lattice that accelerate GTP hydrolysis compared to the free heterodimer as well as confirm that a compacted MT lattice is necessary for hydrolysis, while a more expanded lattice is unable to form the necessary contacts and thereby hydrolyze GTP. Microtubules (MTs) are large cytoskeletal polymers, composed of αβ-tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice versa. Depolymerization is coupled with hydrolysis of guanosine triphosphate (GTP) within β-tubulin. Hydrolysis is favored in the MT lattice compared to a free heterodimer with an experimentally observed rate increase of 500- to 700-fold, corresponding to an energetic barrier lowering of 3.8 to 4.0 kcal/mol. Mutagenesis studies have implicated α-tubulin residues, α:E254 and α:D251, as catalytic residues completing the β-tubulin active site of the lower heterodimer in the MT lattice. The mechanism for GTP hydrolysis in the free heterodimer, however, is not understood. Additionally, there has been debate concerning whether the GTP-state lattice is expanded or compacted relative to the GDP state and whether a “compacted” GDP-state lattice is required for hydrolysis. In this work, extensive quantum mechanics/molecular mechanics simulations with transition-tempered metadynamics free-energy sampling of compacted and expanded interdimer complexes, as well as a free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. α:E254 was found to be the catalytic residue in a compacted lattice, while in the expanded lattice, disruption of a key salt bridge interaction renders α:E254 less effective. The simulations reveal a barrier decrease of 3.8 ± 0.5 kcal/mol for the compacted lattice compared to a free heterodimer, in good agreement with experimental kinetic measurements. Additionally, the expanded lattice barrier was found to be 6.3 ± 0.5 kcal/mol higher than compacted, demonstrating that GTP hydrolysis is variable with lattice state and slower at the MT tip.
登录
查看更多内容
影响因子：
4.4
作者：
Andricioaei, I;Straub, JE
通讯作者：
Straub, JE
DOI：
10.1073/pnas.2114994119
发表时间：
2022-01-11
影响因子：
11.1
作者：
LaFrance BJ;Roostalu J;Henkin G;Greber BJ;Zhang R;Normanno D;McCollum CO;Surrey T;Nogales E
通讯作者：
Nogales E
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：
1.1
作者：
GODBOUT, N;SALAHUB, DR;WIMMER, E
通讯作者：
WIMMER, E
影响因子：
4.8
作者：
Menéndez, M;Rivas, G;Andreu, JM
通讯作者：
Andreu, JM