EF-Tu Translation Factor

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EF-Tu Translation Factor

Holly Byun and Emily Copeland '28

I. Introduction

Elongation Factor Tu (EF-Tu) is a prokaryotic translation factor responsible for delivering aminoacylated tRNA (aa-tRNA) to the ribosomal A-site. EF-Tu is a member of the guanosine triphosphatase (GTPase) family, unique due to its central and highly conserved role in translation, and is dependent on GTP/GDP binding. After binding with GTP, the EF-Tu-GTP complex is active and displays a high binding affinity for aa-tRNAs. EF-Tu delivers aa-tRNAs to the ribosome where the mRNA codon in the A-site is identified by the aa-tRNA. After this process, EF-Tu hydrolyzes GTP to GDP, and the EF-Tu-GDP complex dissociates from the ribosome due to its low binding affinity. Conformational changes are an important part of EF-Tu’s function. EF-Tu is essential for the fidelity of tRNA selection and accurate protein synthesis.

II. General Structure

EF-Tu is a GTPase that enables high-affinity binding and the delivery of aa-tRNAs. Domain 1 contains a GTPase core in addition to the conserved switch I and switch II regions. Domain 1 contains alpha helices and beta strands . Domain 2 is a β-barrel that forms the primary binding pocket for the aminoacylated 3′ end of tRNA, the terminal A76 and the aminoacyl ester insert into this site, and the aminoacyl side chain extends into a large cavity between domains 1 and 2, a feature that allows EF-Tu to tolerate diverse amino acids. Domain 3 contains a second β-barrel responsible for aa-tRNA binding, and is the site of inactive GDP-bound EF-Tu that can be recycled to the active GTP-bound state. Domain 3 extensively contacts the T-stem and contains a kingdom-specific loop . Residue G64 of the T stem forms a hydrogen bond with Gly 391 to help discriminate against initiator and selenocysteine tRNAs.

EF-Tu recognizes universal structural signatures of elongator aa-tRNAs. The CCA-Cys end docks into pockets in domain 2, while the phosphorylated 5′ end is in a positively charged surface depression formed at the intersection of all three domains. This electropositive cradle is shaped by conserved residues. positions the 5′ phosphate relative to the G2 phosphate, and interacts with the 5′ phosphate and with of domain 2 . Together, the three domains of EF-Tu cooperatively generate the Cys-tRNAᶜʸˢ binding site, where the aminoacyl peptide-bond geometry fine-tunes recognition.

III. Switch I

Switch I is found in residues 51 to 64 between the alpha helix and beta sheet of domain 1. Switch I undergoes one of the most dramatic rearrangements during EF-Tu’s transition from the GTP-bound to the GDP-bound state. In the pre-hydrolysis conformation, switch I is coordinated to the γ-phosphate of GTP through T62 . Switch I helps stabilize EF-Tu’s tight grip on the aa-tRNA, particularly around the CCA end and 5′ phosphate contacts. Upon GTP hydrolysis, loss of the γ-phosphate disrupts this coordination and causes switch I to partially open, becoming more solvent-exposed. As EF-Tu moves upon GTP hydrolysis, the loss of the γ-phosphate disrupts Mg²⁺ coordination which causes switch I to open. This initiates the first rotation and loosening of domain 1 away from domains 2 and 3. This results in switch I refolding from an α-helix to form a β-strand, breaking many interdomain hydrogen bonds that previously anchored domain 1 to domains 2 and 3.

This restructuring helps initiate domain 1’s ~90° rotation reshaping the tRNA-binding surface and weakening interactions with aa-tRNA. During domain separation and subsequent rejoining, switch I adopts the extended β-sheet conformation characteristic of the GDP-bound state, contributing to the final locked configuration in which EF-Tu no longer supports tight tRNA binding. Because EF-Tu must rapidly change between these states during translation, the rapid folding transitions in switch I are essential for timely aa-tRNA release.

IV. Switch II

Switch II is found in residues 83 to 100 of Domain 1. Switch II undergoes a critical structural transition that enables EF-Tu’s post-hydrolysis conformation. In the GTP-bound state, switch II is stabilized by a key hydrogen bond between the γ-phosphate of GTP and H85, which is a conserved residue whose position is functionally important. is present in the GTP state to conserve the γ-phosphate while preventing hydrolysis to keep GTP in its active, pre-hydrolysus conformation. This rearrangement favors the GDP-bound conformation. During the separation and rotation of domain 1, switch II continues to remodel the helix containing H85 as it partially unwinds during domain 1 separation. Domain 1 freely rotates toward its GDP-like orientation, then reattaches to domains 2 and 3 in the final conformational rearrangements. Here, final switch-region rearrangements occur that lock EF-Tu into the fully stabilized post-hydrolysis conformation and two conserved residues P83 and Y88 move into new positions. Y88 shifts from being solvent-exposed to inserting into a hydrophobic pocket that only forms once domain 1 has rotated; P83 simultaneously moves into the void left by the departed γ-phosphate. These coordinated motions “lock” EF-Tu into its final GDP-bound architecture. Switch II movements directly influence domain 1 rotation and the weakening of tRNA contacts, and ensure that aa-tRNA release occurs at the correct stage.

V. EF-Tu Targeted Antibiotics

EF-Tu has a central role in the elongation process of protein sysnthesis. EF-Tu cycles between a compact GTP-bound conformation and a looser GDP-bound state with widened interfaces and a central cavity. This structural plasticity makes EF-Tu a major target for antibiotic inhibition of protein synthesis. Four antibiotic families: kirromycin (KIR), enacyloxin (ENX), pulvomycin (PULVO), and GE2270A (GEA), exploit these conformational states through two mechanistic strategies. KIR and ENX act on the EF-Tu·GDP complex on the ribosome, freezing EF-Tu in a GTP-like conformation that holds tightly to aa-tRNA and prevents its release after GTP hydrolysis. Consequently, this blocks formation of the next peptide bond; structurally, both antibiotics bind at the domain-1,3 interface , push domain 1 upward over domains 2 and 3, and destabilize the effector region containing switch I and switch II. KIR achieves additional stabilization by inserting its longer terminal diene “tail” into a nearby hydrophobic pocket that ENX cannot occupy. PULVO and GEA act earlier in the elongation cycle, preventing assembly of the EF-Tu·GTP·aa-tRNA ternary complex. These antibodies bind at partially overlapping but distinct positions across domains 1, 2, and 3. PULVO and GEA sterically block aa-tRNA’s 3′-CCA binding pocket and 5′-phosphate site, while preventing completion of the domain 1 rotation required for EF-Tu activation.

Across all four drug classes, antibiotic binding to domain interfaces communicates through the switch regions to the nucleotide-binding pocket, altering GTPase activity and dramatically increasing EF-Tu’s affinity for GTP. Together, these structural mechanisms illustrate how small molecules modulate a dynamic regulatory GTPase by stabilizing specific conformational states. This highlights EF-Tu as an attractive platform for antibiotic design based on its flexible domain architecture and essential role in translation.

VI. References

Lai J, Ghaemi Z, Luthey-Schulten Z. The Conformational Change in Elongation Factor Tu Involves Separation of Its Domains. Biochemistry. 2017 Nov 14;56(45):5972-5979. doi: 10.1021/acs.biochem.7b00591. Epub 2017 Oct 27. PMID: 29045140.
Parmeggiani A, Nissen P. Elongation factor Tu-targeted antibiotics: four different structures, two mechanisms of action. FEBS Lett. 2006 Aug 21;580(19):4576-81. doi: 10.1016/j.febslet.2006.07.039. Epub 2006 Jul 24. PMID: 16876786.
Poul Nissen, Søren Thirup, Morten Kjeldgaard, Jens Nyborg. The crystal structure of Cys-tRNACys–EF-Tu–GDPNP reveals general and specific features in the ternary complex and in tRNA. Structure Volume 7, Issue 2, 1999, Pages 143-156, ISSN 0969-2126.
Rebecca M. Voorhees, V. Ramakrishnan. 2013. Structural Basis of the Translational Elongation Cycle. Volume 82, Issue 203-236.

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