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Translation, the ribosome dependent synthesis of proteins, is the last step of gene expression. It is targeted by a large number of antibiotics that modulate, in one way or the other, the dynamic properties of the involved biomolecules. Protein synthesis is a highly conserved multi-step process facilitated by a large ribonucleoprotein complex (the ribosome) acting as a biomolecular assembler, converting genetic information provided as RNA transcripts (mRNA) into proteins. This process occurs with high speed and incredible accuracy within any living system (1). As such, accuracy is the key to maintaining the integrity of the genetic information to be reflected in the resulting proteins. The underlying design principles that enable the translation machinery to achieve these performance characteristics is of great interest for a large number of fields and applications, naming antibiotics design and the rational engineering of biomolecular machines just as two examples.

Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli. Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu·EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts.

The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu-GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C.We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

Elongation Factor (EF) Tu is universally conserved and delivers aminoacyl-tRNAs (aatRNAs) to the ribosome. To perform this essential task, EF-Tu binds aa-tRNA in an active GTP-bound state, and GTP hydrolysis is critical for deposition of aa-tRNA into the ribosome. Following each aa-tRNA delivery, nucleotide exchange is required for reactivation of EF-Tu•GDP to EF-Tu•GTP. EF-Tu undergoes a variety of conformational changes during each round of aa-tRNA delivery and reactivation. Here, molecular dynamics simulations in silico and kinetic measurements in vitro demonstrate that structural dynamics of EF-Tu on the sub-nanosecond timescale are correlated with EF-Tu functions on the timescale of seconds. Specifically, structural dynamics of the conserved P-loop and switch II regions are important for nucleotide binding in EF-Tu. Additionally, interactions between domain II of EF-Tu and the small ribosomal subunit, which stimulate GTP hydrolysis on EF-Tu, have the potential to regulate structural dynamics of switch I and switch II.