Elongation Factor Tu’s Nucleotide Binding Is Governed by a Thermodynamic Landscape Unique among Bacterial Translation Factors

Abstract

Insights into the apo Conformation of EF-TuThe first structural insight into the apo conformation of EF-Tu was gleaned from the crystal structure of the EF-Tu•EF-Ts complex 1 . In this structure, switch I of EF-Tu is disordered and the domain arrangement is similar to the GDP conformation. Subsequently, Thirup and co-workers were able to crystalize an EF-Tu•EF-Ts complex with EF-Tu in the closed GTP conformation, indicating that the EF-Tu•EF-Ts complex exhibits conformational flexibility 2 . Our kinetic data reported here provides additional insight in the apo state of EF-Tu and suggests that the free apo state of EF-Tu adopts a unique conformation that is able to recognize GTP and GDP similarly. Similar association and different dissociation activation barriers support that nucleotide binding to this non-discriminatory conformation of EF-Tu is followed by conformational changes of EF-Tu that depend on the presence and absence of the gamma-phosphate in GTP. Therefore, the nucleotide release mechanism is likely the inverse in which a conformational change has to occur prior to nucleotide dissociation. This process would require a unique conformation for EF-Tu•apo and is constant with the reported conformational flexibility observed by Johansen et al and Kavaliauskas et al [3][4] . Considering the entropic landscape of EF-Tu, the EF-Tu•apo conformation is less stable than both EF-Tu•GTP and EF-Tu•GDP (Fig 4). Since EF-Tu employs water coordination to entropically stabilize the GTP conformation it is likely that water coordination stabilizes EF-Tu•GTP and EF-Tu•GDP compared to EF-Tu•apo. If GDP is removed from the structure of EF-Tu•GDP the SASA increases by 109 Å 2 . The difference in SASA between EF-Tu•GTP and EF-Tu•GDP is 1041 Å 2 ( Fig S6B) and since the entropy gap between EF-Tu•GDP and EF-Tu•GTP is similar to the entropy gap between EF-Tu•apo and EF-Tu•GDP ( Fig 4C) it is likely that SASA alone does not explain the entropy of the EF-Tu•apo conformation. Therefore, EF-Tu•apo cannot merely be a similar conformation to EF-Tu•GDP without nucleotide, but has to be a unique conformation. Another possible explanation for the entropy of EF-Tu•apo is that this state is less flexible. This is unlikely as switch I is disordered in the EF-Tu•EF-Ts crystalized complex from Kawashima et al 1 . If this is the conformation that switch I adopts in the apo state then it is likely going to be more flexible. However, this does agree with the observation that EF-Tu•apo coordinates more water molecules, as a disordered switch I would have a larger SASA. Since EF-Tu•apo is less entropically stable compared to EF-Tu•GTP or EF-Tu•GDP which cannot be explained simply by the loss of nucleotide or EF-Tu•apo being less flexible then EF-Tu•apo must adopt a unique conformation. To directly compare the thermodynamic contributions of each nucleotide bound state relative to each other we can use the law of mass-action. However, since GTP contains an additional phosphate compared to GDP mass is not conserved in the kinetic mechanism of EF-Tu nu...

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