title: Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.
abstract: 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.
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Sheng-Wei
authors.initials: SW
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Hsiu-Chi
authors.initials: HC
authors.lastname: Ting
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Lo
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Ting-Yuan
authors.initials: TY
authors.lastname: Wu
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Hung-Wei
authors.initials: HW
authors.lastname: Huang
authors.affiliation: Department of Materials Engineering, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Chia-Jung
authors.initials: CJ
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Jui-Fen Riva
authors.initials: JF
authors.lastname: Chan
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Min-Chieh
authors.initials: MC
authors.lastname: Chuang
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan; Life Science Research Center, Tunghai University, Taichung, Taiwan. Electronic address: howardhsu@thu.edu.tw.
authors.forename: Yuan-Hao Howard
authors.initials: YH
authors.lastname: Hsu
mesh_terms: D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006150:Guanine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D055672:Static Electricity; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D006150:Guanine Nucleotides; D010455:Peptides; D006153:Guanosine Diphosphate; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; D020764:cdc42 GTP-Binding Protein
keywords: Cdc42; GTP activation; H/D exchange; HDXMS; Mass spectrometry; Small G-protein