Lu Shaoyong, Jang Hyunbum, Nussinov Ruth, Zhang Jian
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200025, China.
Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Sci Rep. 2016 Feb 23;6:21949. doi: 10.1038/srep21949.
Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4B(WT)-GTP/GDP) catalytic domain, the K-Ras4B(WT)-GTP-GAP complex, and the mutants (K-Ras4B(G12C/G12D/G12V)-GTP/GDP, K-Ras4B(G13D)-GTP/GDP, K-Ras4B(Q61H)-GTP/GDP) and their complexes with GAP. In addition, we simulated 'exchanged' nucleotide states. These comprehensive simulations reveal that in solution K-Ras4B(WT)-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4B(G12C/G12D)-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.
Ras介导细胞增殖、存活和分化。K-Ras4B中的突变主要发生在G12、G13和Q61残基处。尽管所有这些突变都会损害GAP辅助的GTP→GDP水解，但K-Ras4B在人类癌症中的突变频率各不相同。在这里，我们旨在弄清楚它们的机制和不同的致癌性。我们总共对野生型K-Ras4B（K-Ras4B(WT)-GTP/GDP）催化结构域、K-Ras4B(WT)-GTP-GAP复合物以及突变体（K-Ras4B(G12C/G12D/G12V)-GTP/GDP、K-Ras4B(G13D)-GTP/GDP、K-Ras4B(Q61H)-GTP/GDP）及其与GAP的复合物进行了6.4微秒的分子动力学模拟。此外，我们还模拟了“交换”的核苷酸状态。这些全面的模拟表明，在溶液中K-Ras4B(WT)-GTP以两种构象存在，即活性构象和非活性构象。致癌突变在K-Ras4B-GTP中差异性地引发从非活性到活性的构象转变；在K-Ras4B(G12C/G12D)-GDP中，它们会暴露结合的核苷酸，这有利于GDP与GTP的交换。这些机制可能有助于阐明K-Ras4B驱动的癌症中不同的突变统计情况。交换核苷酸模拟表明，从GTP到GDP的构象转变比从GDP到GTP的交换更容易。重要的是，GAP不仅提供其R789精氨酸指，还稳定催化活性构象并预组织催化残基Q61；突变会干扰R789/Q61的组织，损害GAP介导的GTP水解。总之，我们的模拟有助于为癌症中最具致癌性的蛋白质之一的关键突变事件提供机制解释。
