G-domain dimerization orchestrates the tRNA wobble modification reaction in the MnmE/GidA complex

Area of Science:

• Biochemistry
• Molecular Biology
• Mitochondrial Biology

Background:

• MnmE and GidA proteins modify specific tRNAs, a process vital for cellular function.
• Defects in human orthologs are linked to mitochondrial diseases.
• MnmE functions as a GTP-dependent G protein activated by dimerization.

Purpose of the Study:

• To elucidate the mechanism of MnmE/GidA complex formation and its role in tRNA modification.
• To investigate the structural and functional requirements for MnmE/GidA activity.
• To establish the in vitro and in vivo relevance of the MnmE/GidA complex in cellular respiration.

Main Methods:

• Structural modeling to predict complex interfaces.
• Site-directed mutagenesis to disrupt complex formation and communication.
• In vitro tRNA modification assays.
• Yeast models to assess in vivo function and respiratory deficiency.

Main Results:

• Complex formation induces conformational changes in MnmE, promoting G-domain dimerization and co-stimulating GTP hydrolysis.
• GidA binding affinity is regulated by MnmE's G-domain dimerization state.
• The MnmE/GidA complex, not sequential action, is essential for in vitro glycine incorporation into tRNA.
• GTP hydrolysis, active GTPase, intact MnmE/GidA communication, and G-domain dimerization are critical for in vivo function.

Conclusions:

• The MnmE/GidA complex functions as a single unit, with GTP hydrolysis and G-domain dimerization being essential for tRNA modification.
• Disruptions in MnmE/GidA complex integrity or function lead to mitochondrial dysfunction and respiratory deficiency in yeast.
• This study provides critical insights into the molecular mechanisms underlying tRNA modification and its link to mitochondrial health.