Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: Two different nucleotide states in one crystal

Abstract

The 2.5 Å 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-β-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·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.