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Mycobacterial Methionine Aminopeptidase Type 1c Moonlights as an Anti-association Factor on the 30S Ribosomal Subunit
Aneek Banerjee1, Krishnamoorthi Srinivasan1, Jayati Sengupta1
1Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
Abstract:
Methionine aminopeptidase (MetAP) is a vital metalloprotease that plays a crucial role in protein synthesis by binding to the 70S ribosome at the peptide exit tunnel and removing the N-terminal methionine from nascent polypeptide chains. In Escherichia coli, a single subclass of type 1 MetAP is present, whereas mycobacteria possess two subclasses, MetAP1a and MetAP1c. The key difference between these two is the presence of an additional 40 amino acid-long N-terminal extension in MetAP1c, which may contribute to distinct functional properties. In this study, we have uncovered a previously unrecognized "moonlighting" function of MetAP1c in mycobacteria. Interestingly, our results show that MetAP1c expression is specifically enhanced during the stationary phase of bacterial growth. Moreover, we identify a unique interaction between MetAP1c and the 30S ribosomal subunit, revealing its distinctive affinity for the small subunit. A 4.7 Å cryo-EM map of the Mycobacterium smegmatis MetAP1c-30S subunit complex demonstrates for the first time that MetAP1c binds at the inter-subunit face of the 30S subunit head region. The binding of MetAP1c induces conformational changes in the 30S subunit, impairing its ability to associate with the 50S subunit, thus imparting an anti-association property to MetAP1c. To further understand the role of the N-terminal extension, we constructed two mutant variants of MetAP1c, which confirmed its critical involvement in this moonlighting function. This anti-association activity of MetAP1c is likely one of the energy conservation mechanisms in mycobacteria where MetAP1c is involved in translation down regulation during stationary phase.
Insights
Mycobacterial methionine aminopeptidase 1c (MetAP1c) has a novel role in regulating protein synthesis. This enzyme binds the 30S ribosomal subunit, preventing ribosome assembly and downregulating translation during stationary phase.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Methionine aminopeptidase (MetAP) enzymes remove N-terminal methionine from proteins during synthesis.
- Mycobacteria have two MetAP subclasses, MetAP1a and MetAP1c, with MetAP1c possessing a unique N-terminal extension.
- The function of MetAP1c's N-terminal extension and its role beyond methionine removal are not well understood.
Purpose of the Study:
- To investigate the potential "moonlighting" functions of MetAP1c in mycobacteria.
- To elucidate the structural basis and functional implications of MetAP1c's interaction with the ribosome.
- To understand the role of MetAP1c in bacterial adaptation during stationary phase.
Main Methods:
- Cryo-electron microscopy (cryo-EM) to determine the structure of the MetAP1c-30S ribosomal subunit complex.
- Site-directed mutagenesis to assess the role of MetAP1c's N-terminal extension.
- Analysis of MetAP1c expression levels during different growth phases.
Main Results:
- MetAP1c expression is upregulated during the stationary phase of bacterial growth.
- MetAP1c directly binds to the 30S ribosomal subunit at the inter-subunit face of the head region.
- MetAP1c binding induces conformational changes in the 30S subunit, inhibiting its association with the 50S subunit, thus downregulating translation.
- The N-terminal extension of MetAP1c is crucial for its anti-association activity.
Conclusions:
- MetAP1c exhibits a novel moonlighting function as a ribosomal anti-association factor.
- This activity contributes to translational regulation and energy conservation in mycobacteria during stationary phase.
- MetAP1c's interaction with the 30S subunit represents a unique mechanism for controlling protein synthesis.
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