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Nucleotide binding to the G12V‐mutant of Cdc42 investigated by X‐ray diffraction and fluorescence spectroscopy: Two different nucleotide states in one crystal
Author(s) -
Rudolph Markus Georg,
Wittinghofer Alfred,
Vetter Ingrid Renate
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.4.778
Subject(s) - crystallography , guanosine diphosphate , chemistry , intramolecular force , crystallization , molecule , nucleotide , stereochemistry , guanine , biochemistry , organic chemistry , gene
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/Mg 2+ and GDPNH 2 (guanosine‐5′‐diphospho‐β‐amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg 2+ , while the other has bound GDPNH 2 without a Mg 2+ 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 GDPNH 2 vs. GDP. The amino group of GDPNH 2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg 2+ 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 GDPNH 2 . 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.