Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal.
The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.
Rudolph
M G
MG
Max-Planck Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Dortmund, Germany.
Wittinghofer
A
A
Vetter
I R
IR
D018797:Cell Cycle Proteins / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014961:X-Ray Diffraction / Q000379:methods*; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D018631:ras Proteins / Q000737:chemistry; D020742:rhoA GTP-Binding Protein
D018797:Cell Cycle Proteins; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein; D008274:Magnesium