An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function

JOURNAL OF MOLECULAR BIOLOGY

卷:430

Article

Ruiz-Partida, Rafael¹  Prado, Silvia¹  Villarroya, Magda¹  Velazquez-Campoy, Adrian^2,3,4,5,6  Bravo, Jeronimo⁷  Armengod, M. -Eugenia^1,8 

[1] Ctr Invest Principe Felipe, Valencia 46012, Spain

[2] Univ Zaragoza, Inst Biocomputat & Phys Complex Syst BIFI, Joint Units IQFR CSIC BIFI & GBsC CSIC BIFI, Zaragoza 50018, Spain

[3] Univ Zaragoza, Dept Biochem & Mol & Cell Biol, Zaragoza 50018, Spain

[4] Aragon Inst Hlth Res IIS Aragon, Zaragoza 50009, Spain

[5] Biomed Res Networking Ctr Liver & Digest Dis CIBE, Madrid 28029, Spain

[6] Govt Aragon, Fdn ARAID, Zaragoza 50018, Spain

[7] CSIC, Inst Biomed Valencia, Valencia 46010, Spain

[8] Biomed Res Networking Ctr Rare Dis CIBERER Node 7, Valencia, Spain

关键词: flavoenzymes;    interprotomer disulfide bridges;    MnmE;    MTO1;    sterile alpha motif;   

DOI  :  10.1016/j.jmb.2018.05.035

来源: Elsevier

【 摘 要 】

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. (C) 2018 The Authors. Published by Elsevier Ltd.

【 授权许可】

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