The Structure of the EF‐Tu · GDP · Me2+ Complex

Abstract

The structure of the MgGDP complex at the active site of elongation factor (EF-Tu) has been investigated by using phosphorothioate analogs of GDP in the absence and presence of various metal ions, electron para-magnetic resonance (EPR) and nuclear magnetic resonance (NMR) measurements. The high stereoselectivity of EF-Tu for the diastereomers of guanosine 5′-0-(1-thiodiphosphate) (GDP[αS]) is independent of the nature of the metal ion and is caused by the interaction of the protein with the α-phosphate of GDP. By using GDP analogs where the oxygens at either thea-phosphate or thep-phosphate have been selectively labelled with ¹⁷O and measuring their effect on the EPR spectrum of EF-Tu-bound manganese we are able to show that only the β-phosphate of GDP is coordinated to the metal ion in the EF-Tu. Me²⁺. GDPcomplex. ³¹P-NMR studies on GDP and guanosine 5′-0-(2-thiodiphosphate) (GDP[βS]) bound to EF-Tu indicate that in the EF-Tu Me²⁺ GDP complex Mg²⁺ interacts more strongly with the β-phosphate than with the α-phosphate. Together with binding studies using GDP[βS] our NMR results also indicate that the protein is complexed to the β-phosphorous of GDP via two oxygens.