ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery
- University of Bristol, United Kingdom
- King's College London, United Kingdom
- University of St Andrews, United Kingdom
- University of South Bohemia in Ceske Budejovice, Czech Republic
Abstract
Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide [Allen et al. eLife 2016]. Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogen-deuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.
Data availability
All data generated during this study are included in the Figures of the mansucript. EPR data is available at https://doi.org/10.17630/0fedaeec-7e27-4876-a6d1-cda2d3a6799c.
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EPR data from ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinerydoi:10.17630/0fedaeec-7e27-4876-a6d1-cda2d3a6799c.
Article and author information
Author details
William John Allen
Funding
Biotechnology and Biological Sciences Research Council (BB/M003604/1)
- Robin A Corey
- William John Allen
- Ian Collinson
Wellcome (104632)
- William John Allen
- Ian Collinson
Royal Society (University Research Fellowship)
- Janet E Lovett
Wellcome (109854/Z/15/Z)
- Zainab Ahdash
- Argyris Politis
Wellcome (099149/Z/12/Z)
- Anokhi Shah
- Janet E Lovett
European Regional Development Fund (CZ.02.1.01/0.0/0.0/15_003/0000441)
- Tomas Fessl
Engineering and Physical Sciences Research Council (ep/m508214/1)
- Anokhi Shah
Biotechnology and Biological Sciences Research Council (BB/I008675/1)
- Robin A Corey
- William John Allen
- Ian Collinson
Biotechnology and Biological Sciences Research Council (BB/N015126/1)
- Robin A Corey
- William John Allen
- Ian Collinson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Corey 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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ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery
eLife 8:e41803.
https://doi.org/10.7554/eLife.41803
Further reading
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
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- Biochemistry and Chemical Biology
- Computational and Systems Biology
Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.
