The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity
- University of Birmingham, United Kingdom
- Heidelberg University, Germany
- University of Heidelberg, Germany
Abstract
In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA can bind ribosomes and recognise nascent substrate proteins, but the molecular mechanism of recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.
Data availability
X-ray crystallography data are deposited in PDB under accession code 6GOX.Small-angle x-ray scattering data are deposited in SASBDB under accession codes SASDDY9, SASDDZ9 and SASDE22.
Article and author information
Author details
Funding
Biotechnology and Biological Sciences Research Council (BB/L019434/1)
- Mohammed Jamshad
- Damon Huber
Biotechnology and Biological Sciences Research Council (BB/P009840/1)
- Timothy J Knowles
- Gareth W Hughes
Biotechnology and Biological Sciences Research Council (MIBTP)
- Max Wynne
Deutsche Forschungsgemeinschaft (FOR 1805)
- Günter Kramer
- Bernd Bukau
Deutsche Forschungsgemeinschaft (SFB 638)
- Günter Kramer
- Bernd Bukau
Wellcome (099266/Z/12/Z)
- Fiyaz Mohammed
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Jamshad et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity
eLife 8:e48385.
https://doi.org/10.7554/eLife.48385
Further reading
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
