Journal of Molecular Biology
Volume 430, Issue 17, 17 August 2018, Pages 2822-2842
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An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function

https://doi.org/10.1016/j.jmb.2018.05.035Get rights and content
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Highlights

  • The architecture of the tRNA modification complex MnmE–MnmG is currently unknown.

  • The MnmG form I and II structures differ in their dimerization interfaces.

  • Form I is biologically active and oscillates between open and closed conformations.

  • Form I conformational dynamics regulates the access of MnmE to the MnmG active center.

  • Form I conformational dynamics regulates the access of tRNA to the MnmG active center.

Abstract

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA–protein and protein–protein interactions.

Abbreviations

AaMnmG
MnmG from Aquifex aeolicus
EcMnmG
MnmG from Escherichia coli
CtMnmG
MnmG from Chlorobium tepidum
U34
uridine 34
NRE
native red electrophoresis
EMSA
electrophoretic mobility shift assay
ITC
isothermal titration calorimetry
cmnm
carboxymethylaminomethyl group
nm
aminomethyl group
SAM
sterile alpha motif

Keywords

flavoenzymes
interprotomer disulfide bridges
MnmE
MTO1
sterile alpha motif

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