Yang Sheng-Wei, Ting Hsiu-Chi, Lo Yi-Ting, Wu Ting-Yuan, Huang Hung-Wei, Yang Chia-Jung, Chan Jui-Fen Riva, Chuang Min-Chieh, Hsu Yuan-Hao Howard
Department of Chemistry, Tunghai University, Taichung, Taiwan.
Department of Materials Engineering, National Chung Hsing University, Taichung, Taiwan.
Biochim Biophys Acta. 2016 Jan;1864(1):42-51. doi: 10.1016/j.bbapap.2015.10.007. Epub 2015 Nov 2.
Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.
Cdc42调节与细胞分裂相关的信号通路。Cdc42失调可导致癌症、心血管疾病和神经退行性疾病。GTP诱导的激活机制在Cdc42的活性和生物学功能中起重要作用。P环、开关I和开关II是调节Cdc42酶活性的关键区域。我们应用酰胺氢/氘交换结合液相色谱质谱法（HDXMS）来研究空载Cdc42在结合GDP、GTP和GMP-PCP后的动态变化。天然底物GTP导致P环和开关II中的氘化显著降低，开关I中的氘化有适度变化，而在远离活性位点的α7螺旋中的氘化有显著变化。GTP结合对氢/氘交换的影响与其不可水解类似物GMP-PCP相似。HDXMS结果表明，GTP结合阻断了活性位点的溶剂可及性，导致活性位点周围氢/氘交换率降低，并进一步引发构象变化，导致远端α7螺旋处氢/氘交换率急剧下降。比较空载Cdc42、Cdc42-GDP和Cdc42-GMP-PCP三种激活状态下的氘化水平，空载Cdc42具有最灵活的结构，鸟嘌呤核苷酸结合可使其稳定。Cdc42-GDP的氢/氘交换率介于结合GMP-PCP和空载形式之间，但更接近结合GMP-PCP的形式。我们的结果表明，Cdc42的激活是一个涉及P环、开关II和α7螺旋以实现结构稳定的构象变化过程。
