Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry

• Published in: Biochimica et biophysica acta Vol. 1864; no. 1; pp. 42 - 51
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2016
• Subjects: Amino Acid Sequence; Cdc42; cdc42 GTP-Binding Protein - chemistry; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Deuterium Exchange Measurement - methods; GTP activation; Guanine Nucleotides - chemistry; Guanine Nucleotides - metabolism; Guanosine Diphosphate - chemistry; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - analogs & derivatives; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; H/D exchange; HDXMS; Humans; Mass spectrometry; Models, Molecular; Molecular Sequence Data; Peptides - chemistry; Peptides - metabolism; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Small G-protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Static Electricity; P-loop; Small G-protein; HDXMS; GMP-PCP; GTP activation; H/D exchange; Mass spectrometry; ODT; Cdc42
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2015.10.007

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. •HDXMS revealed the dynamic changes of apo-Cdc42 upon guanine nucleotide binding.•Guanine nucleotide binding decreased deuteration in P-loop, Switch II and α7 helix.•GTP/GDP binding blocked the solvent accessibility in the active site.•GTP/GDP binding induced a conformational change at the remote α7 helix.