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Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
Conserved Conformational Changes in the Regulation of Mycobacterium tuberculosis MazEF-mt1
Ran Chen1, Jie Zhou1, Runlin Sun1
1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, The Sun Yat-Sen University, Guangzhou, Guangdong 510006, People's Republic of China.
Abstract:
Toxin-antitoxin (TA) systems, which regulate many important cellular processes, are abundantly present in prokaryotic organisms. MazEF is a common type of TA system implicated in the formation of "persisters cells" of the pathogen Mycobacterium tuberculosis, which contains 10 such systems. However, the exact function and inhibition mode of each MazF protein are not quite understood. Here, we report four high-resolution crystal structures of MazF-mt1 in various forms, including one in complex with MazE-mt1. The toxin displayed two unique interlocked loops that allow the formation of a tight dimer. These loops would open upon interacting with the MazE-mt1 antitoxin mediated by the last two helices of MazE-mt1. With our structure-based design, a mutant that could bind to the antitoxin with an enhanced affinity was produced. Combined crystallographic and biochemical studies further revealed that the binding affinity of MazE-mt1 to MazF-mt1 was mainly attributed to its α3 helical region, while the terminal helix η1 contributes very little or even negatively to the association of the pair, in stark contrast to the MazEF-mt9 system. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F-mt1 TA pair, which may reflect the functional differences between different TA systems.
Insights
Toxin-antitoxin systems regulate cellular processes. Researchers elucidated the MazE/F-mt1 structure, revealing how the antitoxin inhibits the toxin, offering insights into Mycobacterium tuberculosis persister cell formation.
Area of Science:
- Microbiology
- Structural Biology
- Molecular Biology
Background:
- Toxin-antitoxin (TA) systems are crucial for prokaryotic cellular regulation.
- The MazEF system in *Mycobacterium tuberculosis* contributes to persister cell formation, but individual MazF protein functions remain unclear.
- Understanding TA system dynamics is key to targeting bacterial persistence.
Purpose of the Study:
- To elucidate the structural basis of MazF-mt1 toxin inhibition by MazE-mt1 antitoxin.
- To investigate the molecular interactions governing the MazE-mt1/MazF-mt1 complex formation.
- To explore structure-based strategies for modulating TA system interactions.
Main Methods:
- High-resolution crystal structure determination of MazF-mt1 and its complex with MazE-mt1.
- Structure-guided protein engineering to create a high-affinity MazF-mt1 mutant.
- Biochemical assays to quantify binding affinities and analyze interaction interfaces.
Main Results:
- Four high-resolution crystal structures of MazF-mt1 revealed unique interlocked loops facilitating dimerization.
- Complex formation with MazE-mt1 induced conformational changes in MazF-mt1, mediated by MazE-mt1's C-terminal helices.
- A structure-designed mutant exhibited enhanced binding to MazE-mt1, with the α3 helix of MazE-mt1 being the primary interaction site.
Conclusions:
- The study provides detailed structural insights into the MazE/F-mt1 toxin-antitoxin interaction and inhibition mechanism.
- The findings highlight distinct binding contributions of MazE-mt1 helices compared to other MazEF systems.
- Understanding these molecular details may inform strategies against *Mycobacterium tuberculosis* persistence.
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