SURFS and AlphaFold Reveal Ribosome Footprint Shift Caused by EF-Tu D81 Mutation

Protein synthesis relies on accurate mRNA decoding by tRNA, a process guided by EF-Tu. We investigated how mutations at a conserved residue, aspartate 81, affect EF-Tu function using GTPase assays, AlphaFold modeling, and quantum-sensing-based super-resolution force spectroscopy (SURFS). All D81 variants retained GTPase activity but impaired tRNA release, revealed by sub-nucleotide ribosome footprinting. AlphaFold3 modeling suggests that D81 mutations disrupt magnesium coordination and interaction with the sarcin–ricin loop in the GTP-bound state. AlphaFold2-based sequence–structure analysis indicates that D81 anchors coevolutionary constraints, and its mutation enables cryptic structural variation. These results show how a single conserved residue links catalytic coordination, allosteric communication, and evolutionary constraint, offering mechanistic insight into translation fidelity and demonstrating the utility of an unconventional force spectroscopy in probing ribosome dynamics.

• Publication year

  2025

• Venue

  bioRxiv

• Publication date

  2025-06-07

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1101/2025.06.05.657321PMID 40501600PMCID 12157627
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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