Mechanism-Based Redesign of GAP to Activate Oncogenic Ras.
Mechanism-Based Redesign of GAP to Activate Oncogenic Ras.
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DOI：
10.1021/jacs.3c04330
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发表时间：
2023-09-20
影响因子：
15
通讯作者：
Rosta E
中科院分区：
文献类型：
--
作者：
Berta D;Gehrke S;Nyíri K;Vértessy BG;Rosta E
Ras GTP酶在细胞信号传导途径中起着至关重要的作用。大约三分之一的癌细胞系中存在Ras基因突变，并且通常与不利的临床预后相关。热点残基Gly 12、Gly 13和Gln 61覆盖了97%的致癌突变，这损害了Ras中的酶活性。利用QM/MM自由能计算，我们提出了野生型Ras.GAP复合物催化GTP水解的两步机理。我们发现，催化水的去质子化发生通过Gln 61作为一个短暂的布朗斯特碱。我们还使用QM/MM最小化确定了关键致癌Ras突变体G12 D和G12 C的反应曲线，与实验观察到的催化活性损失相匹配，从而验证了我们的反应机制。使用优化的反应路径，我们设计了一个快速和准确的程序来设计激活G12 D Ras的GAP突变体。我们替换了活性位点附近的GAP残基，并确定了190个单突变体的激活屏障。我们还通过快速预测屏障高度来构建用于超快筛选的机器学习，并对单突变和双突变进行测试。这项工作表明，通过QM/MM反应路径优化可以实现快速准确的筛选，以设计具有更高催化活性的蛋白质序列。预计几个GAP突变将重新启动致癌G12 D中的催化，通过激活酶活性而不是抑制来克服异常Ras驱动的信号转导，提供了一种有前途的途径。概述的计算筛选协议是容易适用于类似的配体和辅因子的设计。
Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Brønsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously.
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影响因子：
8.8
作者：
Jácome AA;Vreeland TJ;Johnson B;Kawaguchi Y;Wei SH;Nancy You Y;Vilar E;Vauthey JN;Eng C
通讯作者：
Eng C
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影响因子：
8.4
作者：
Arafet K;Serrano-Aparicio N;Lodola A;Mulholland AJ;González FV;Świderek K;Moliner V
通讯作者：
Moliner V
影响因子：
4
作者：
Hobbs, G. Aaron;Der, Channing J.;Rossman, Kent L.
通讯作者：
Rossman, Kent L.
影响因子：
3.8
作者：
Aran, Veronica;Heringer, Manoela;Neto, Vivaldo Moura
通讯作者：
Neto, Vivaldo Moura
影响因子：
3.7
作者：
de Lange, Mark J.;Razzaq, Lubna;van der Velden, Pieter A.
通讯作者：
van der Velden, Pieter A.