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Abstract

Molecular switches such as GTPases are powerful devices turning "on" or "off" biomolecular processes at the core of critical biological pathways. To develop molecular switches de novo, an intimate understanding of how they function is required. Here we investigate the thermodynamic parameters that define the nucleotide-dependent switch mechanism of elongation factor (EF) Tu as a prototypical molecular switch. EF-Tu alternates between GTP- and GDP-bound conformations during its functional cycle, representing the "on" and "off' states, respectively. We report for the first time that the activation barriers for nucleotide association are the same for both nucleotides, suggesting a guanosine nucleoside or ribose-first mechanism for nucleotide association. Additionally, molecular dynamics (MD) simulations indicate that enthalpic stabilization of GDP binding compared to GTP binding originates in the backbone hydrogen bonding network of EF-Tu. In contrast, binding of GTP to EF-Tu is entropically driven by the liberation of bound water during the GDP- to GTP-bound transition. GDP binding to the apo conformation of EF-Tu is both enthalpically and entropically favored, a feature unique among translational GTPases. This indicates that the apo conformation does not resemble the GDP-bound state. Finally, we show that antibiotics and single amino acid substitutions can be used to target specific structural elements in EF-Tu to redesign the thermodynamic landscape. These findings demonstrate how, through evolution, EF-Tu has fine-tuned the structural and dynamic features that define nucleotide binding, providing insight into how altering these properties could be exploited for protein engineering.