The mechanism of SecA-dependent substrate recognition and delivery in Escherichia coli
Lead Research Organisation:
University of Birmingham
Department Name: Sch of Biosciences
Abstract
The ultimate goal of our research is to understand how proteins are transported across the cytoplasmic membrane (CM) in bacteria starting with synthesis of a protein in the cytoplasm and ending with its maturation on the other side of the CM.
The CM plays a critical role in the definition of a cell and thus our definition of life. Cells are sometimes described as "bags of proteins", in which the CM is the "bag". It divides the contents of the cell (the cytoplasm) from the extracellular milieu, and it protects the cell from changes in the environment. Without the bag, there is no cell.
However, the CM also poses a fundamental problem. A large number of proteins carry out (often essential) functions outside the cell. In the bacterium Escherichia coli, these proteins comprise ~30% of all proteins synthesized. Protein synthesis occurs exclusively in the cytoplasm, but proteins cannot cross the CM on their own. Thus, the cell has evolved a number of complex machineries to transport proteins across the CM. Of these, the Sec machinery is responsible for the vast majority of protein export. The central component of the Sec machinery is a universally conserved channel in the CM. In bacteria, most proteins are transported through this channel by a pump protein, SecA, only after they have been fully (or nearly fully) synthesized.
Recognition of protein substrates by the Sec machinery is rapid, efficient, and very accurate, but despite decades of research, the process by which substrate proteins are recognized remains an unsolved problem. However, recent research by DH sheds new light on this old problem. This research suggests that SecA itself recognizes substrate proteins, and recognition occurs as the substrate protein is still being made-long before transport across the CM commences. The research described in this proposal aims to understand: (i) how substrate proteins are recognized by SecA, (ii) how substrate proteins are subsequently delivered to the channel in the CM, and (iii) how the timing of recognition and delivery affects the subsequent maturation of the substrate protein on the other side of the CM.
Previous research in this area has been directly applied in the development of new tools for biotechnology. For example, during his PhD research, DH identified a set of signals that could target proteins to be transported across the CM by a second parallel pathway that is more efficient than the SecA-dependent pathway, and these signals have been widely used to secrete proteins that are otherwise refractory to protein transport. However, export by this parallel pathway has a number of significant limitations, including low protein expression levels, cellular toxicity, and in some cases, defective protein maturation. Greater insight into the mechanism of SecA-dependent transport could lead to improved methods for protein production.
This research also has the potential to contribute to new approaches for combating bacterial infections. Some bacteria such as E. coli contain a second membrane outside the cell that serves as a barrier to many antimicrobial compounds, and the proper maturation of transported proteins is important for maintaining the integrity this barrier. Thus, the strong link between route by which proteins are delivered to the channel in the CM and the maturation of the transported protein could be exploited to increase the sensitivity of these bacteria to many otherwise useless antimicrobial compounds.
Finally, many of the techniques developed in this grant can be adapted for use in other studies. For example, the high-throughput sequencing (HTS) methods developed to examine the structure of SecA-ribosome complex can be used to examine the interaction of other proteins with other large RNA or DNA molecules, and the use of HTS to identify novel pathways involved in OM biogenesis can be extended to research into other biological pathways.
The CM plays a critical role in the definition of a cell and thus our definition of life. Cells are sometimes described as "bags of proteins", in which the CM is the "bag". It divides the contents of the cell (the cytoplasm) from the extracellular milieu, and it protects the cell from changes in the environment. Without the bag, there is no cell.
However, the CM also poses a fundamental problem. A large number of proteins carry out (often essential) functions outside the cell. In the bacterium Escherichia coli, these proteins comprise ~30% of all proteins synthesized. Protein synthesis occurs exclusively in the cytoplasm, but proteins cannot cross the CM on their own. Thus, the cell has evolved a number of complex machineries to transport proteins across the CM. Of these, the Sec machinery is responsible for the vast majority of protein export. The central component of the Sec machinery is a universally conserved channel in the CM. In bacteria, most proteins are transported through this channel by a pump protein, SecA, only after they have been fully (or nearly fully) synthesized.
Recognition of protein substrates by the Sec machinery is rapid, efficient, and very accurate, but despite decades of research, the process by which substrate proteins are recognized remains an unsolved problem. However, recent research by DH sheds new light on this old problem. This research suggests that SecA itself recognizes substrate proteins, and recognition occurs as the substrate protein is still being made-long before transport across the CM commences. The research described in this proposal aims to understand: (i) how substrate proteins are recognized by SecA, (ii) how substrate proteins are subsequently delivered to the channel in the CM, and (iii) how the timing of recognition and delivery affects the subsequent maturation of the substrate protein on the other side of the CM.
Previous research in this area has been directly applied in the development of new tools for biotechnology. For example, during his PhD research, DH identified a set of signals that could target proteins to be transported across the CM by a second parallel pathway that is more efficient than the SecA-dependent pathway, and these signals have been widely used to secrete proteins that are otherwise refractory to protein transport. However, export by this parallel pathway has a number of significant limitations, including low protein expression levels, cellular toxicity, and in some cases, defective protein maturation. Greater insight into the mechanism of SecA-dependent transport could lead to improved methods for protein production.
This research also has the potential to contribute to new approaches for combating bacterial infections. Some bacteria such as E. coli contain a second membrane outside the cell that serves as a barrier to many antimicrobial compounds, and the proper maturation of transported proteins is important for maintaining the integrity this barrier. Thus, the strong link between route by which proteins are delivered to the channel in the CM and the maturation of the transported protein could be exploited to increase the sensitivity of these bacteria to many otherwise useless antimicrobial compounds.
Finally, many of the techniques developed in this grant can be adapted for use in other studies. For example, the high-throughput sequencing (HTS) methods developed to examine the structure of SecA-ribosome complex can be used to examine the interaction of other proteins with other large RNA or DNA molecules, and the use of HTS to identify novel pathways involved in OM biogenesis can be extended to research into other biological pathways.
Technical Summary
SecA-mediated transport is the primary pathway for transporting periplasmic and outer membrane proteins across the cytoplasmic membrane (CM) in E. coli. Very little is known about how this pathway recognizes newly synthesized substrate proteins, but recent research by DH and colleagues indicates that substrate protein recognition is cotranslational and is driven by the interaction of the ATPase SecA with the ribosome. The goal of this research is to determine the mechanism of cotranslational substrate recognition by SecA and subsequent delivery to the CM-embedded translocation machinery. To this end, we will probe the structure of the SecA-ribosome complex in vitro using site-specific crosslinking to map the interaction of SecA with ribosomal proteins and RNA footprinting to map its interaction with the ribosomal RNA. In addition, we will combine RNA footprinting with high-throughput sequencing (HTS) to gain a very detailed map of the SecA-binding surface on the rRNA. We will use fluorescence-based approaches, such as FRET and fluorescence anisotropy, to examine the kinetic and steady-state interactions of SecA with the ribosome, nascent substrate protein, SecB, and trigger factor (TF) in vitro. We will also use fluorescence-based methods to examine the conformational changes within SecA upon interaction with the ribosome and nascent polypeptides, and we will determine the x-ray crystal structure of a variant of SecA that is locked in the substrate-bound conformation. In addition, we will use standard genetic and molecular biology approaches to determine how the mechanism of delivery to the CM-embedded translocation machinery can affect subsequent maturation of the substrate protein in vivo. Finally, we will pair HTS with a genetic selection for suppressor mutants in order to develop a high-throughput screen to identify which pathway(s) in the periplasm is affected by altering the mechanism of delivery.
Planned Impact
The research described in this proposal supports to the BBSRC strategic research priority in "Basic biosciences research underpinning health". It provides new insight into a fundamental cellular process: Sec-dependent transport of proteins in bacteria. The primary subject of this study, the protein SecA, is essential for bacterial viability, and altering the mechanism of substrate recognition by SecA and translocation across the cytoplasmic membrane can increase the sensitivity of Escherichia coli to antimicrobials. Thus, this research constitutes "fundamental...studies of...microbial biology leading to potential new antimicrobial drugs and to improvements in both human and animal health" as well as "molecular cell biology...biochemistry and biophysics" that "drive[s] the discovery and validation of new drug targets leading to more effective and/or selective pharmaceuticals". We describe how we will assist in "achiev[ing] a common interface with the pharmaceutical and healthcare industries for more co-ordinated development" in the Pathways to Impact section.
Previous research in this area has led to the development of methods to improve expression of proteins in the E. coli periplasm. For example, pET39 contains the DsbA-Tag(TM), which can increase the expression of recalcitrant proteins in the periplasm. This technology has allowed other groups to manipulate previously intractable proteins using phage display-a commonly used method for selecting for protein-based biopharmaceuticals. The research described in the present proposal has the potential to further improve expression of still intractable proteins in the periplasm for various purposes. Thus, this research "underpin[s] biopharmaceutical development, particularly in areas such as bioprocessing, to improve the manufacture, quality and yield of...complex products, and ultimately to drive down costs".
The expression of proteins on the surface of E. coli is being developed by groups at the University of Birmingham for use in biotechnology and for the production of orally delivered vaccines. Transport across the cytoplasmic membrane by the Sec machinery is the first step in the insertion of proteins into and secretion of proteins across the outer membrane in E. coli and other Gram-negative bacteria, and the mechanism of transport can significantly secretion across the outer membrane. We will therefore work closely with these groups in order to ensure efficient development of these tools for the benefit of biotechnology and health workers.
The development of high-throughput sequencing techniques for the analysis of protein-RNA interactions and multiplex suppressor analysis supports the BBSRC research priority in data-driven biology. The new techniques developed as a part of this research constitute "tools and approaches that are required to underpin and enable modern biological research as it continues to evolve as a data intensive discipline" and involves multidisciplinary partnership with colleagues in information technology at the University of Birmingham and will "provide tools and resources of potential application to broad communities in the biosciences".
Postgraduate and undergraduate students will gain lab experience crucial for their future careers from carrying out related research projects. In addition, the outreach work described in the Pathway to Impact will provide a means to educate and inform the public about BBSRC-funded research.
Previous research in this area has led to the development of methods to improve expression of proteins in the E. coli periplasm. For example, pET39 contains the DsbA-Tag(TM), which can increase the expression of recalcitrant proteins in the periplasm. This technology has allowed other groups to manipulate previously intractable proteins using phage display-a commonly used method for selecting for protein-based biopharmaceuticals. The research described in the present proposal has the potential to further improve expression of still intractable proteins in the periplasm for various purposes. Thus, this research "underpin[s] biopharmaceutical development, particularly in areas such as bioprocessing, to improve the manufacture, quality and yield of...complex products, and ultimately to drive down costs".
The expression of proteins on the surface of E. coli is being developed by groups at the University of Birmingham for use in biotechnology and for the production of orally delivered vaccines. Transport across the cytoplasmic membrane by the Sec machinery is the first step in the insertion of proteins into and secretion of proteins across the outer membrane in E. coli and other Gram-negative bacteria, and the mechanism of transport can significantly secretion across the outer membrane. We will therefore work closely with these groups in order to ensure efficient development of these tools for the benefit of biotechnology and health workers.
The development of high-throughput sequencing techniques for the analysis of protein-RNA interactions and multiplex suppressor analysis supports the BBSRC research priority in data-driven biology. The new techniques developed as a part of this research constitute "tools and approaches that are required to underpin and enable modern biological research as it continues to evolve as a data intensive discipline" and involves multidisciplinary partnership with colleagues in information technology at the University of Birmingham and will "provide tools and resources of potential application to broad communities in the biosciences".
Postgraduate and undergraduate students will gain lab experience crucial for their future careers from carrying out related research projects. In addition, the outreach work described in the Pathway to Impact will provide a means to educate and inform the public about BBSRC-funded research.
Organisations
Publications
Cranford-Smith T
(2018)
The way is the goal: how SecA transports proteins across the cytoplasmic membrane in bacteria.
in FEMS microbiology letters
Cranford-Smith T
(2020)
Iron is a ligand of SecA-like metal-binding domains in vivo.
in The Journal of biological chemistry
Cranford-Smith T
(2019)
Iron is a ligand of SecA-like metal-binding domains in vivo
Huber D
(2017)
SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo.
in Journal of bacteriology
Hughes GW
(2019)
Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system.
in Nature microbiology
Jamshad M
(2019)
The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity.
in eLife
Sen H
(2017)
Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.
in Journal of bacteriology
| Description | The goal of the work in this grant was understand how protein are recognised by the bacterial Sec machinery, which transports newly synthesised proteins across the cytoplamsic membrane. Previous work by DH established that SecA, a conserved and essential component of the bacterial Sec machinery, binds to the ribosome. We hypothesised that SecA binds to the ribosome in order to recognise substrate proteins while they are still being synthesised (i.e. cotranslationally). The aim of the work described in this grant was to investigate molecular mechanism of substrate protein recognition. To this end, we acheive this end, we sought four main objectives: 1. Biochemically define the structure of the SecA-ribosome complex. We sought to use biochemical approaches to refine our model of ribosome bound SecA. We use site-specific crosslinking to understand which parts of SecA contacted different ribosomal proteins. These studies were critical to understanding that SecA undergoes a conformational change when it binds to substrate protein, and they indirectly provided a tool for crosslinking SecA to nascent polypeptides in cells. We reported our results from these studies in Journal of Bacteriology 199 (2), e00622-16 and eLife 8, e48385 2. Determine the dynamics of the different conformational states of SecA. Preliminary work suggested that SecA occupies multiple structural conformations and that these different conformations have differing affinities for substrate proteins and the ribosome. We sought to understand how these played into the mechanism of substrate protein recognition. Our results suggests that there are three different structural conformations of SecA, which we characterised extensively using site-specific crosslinking, x-ray crystallography and small-angle x-ray scattering in eLife 8, e48385. A high-throughput genetic screen described in Journal of Biological Chemistry 295 (21), 7516-7528 suggests these conformational changes could regulate the activity of SecA posttranslationally in response to an as-yet unidentified stress. 3. Determine the mechanism by which SecB and trigger factor (TF) affect the timing of translocation. SecB and TF are known to affect the timing with which new synthesised proteins are delivered to the membrane-embedded Sec machinery. We sought to understand the mechanism underlying these interactions. Results reported in Journal of Bacteriology 199 (2), e00622-16 suggest that SecA and TF can bind to the ribosome and to the same nascent polypeptide simultaneously. These results also suggest that binding of SecB to nascent polypeptides is dependent on SecA in vivo. Further work on the conformational dynamics of SecA reported in eLife 8, e48385 suggest a molecular mechanism. 4. Investigate how the increased level of cotranslational translocation in mutants lacking TF affects OM biogenesis in vivo. TF is a cytoplasmic molecular chaperone, but mutations disrupting TF affect cause newly synthesised substrate proteins to begin translocation while they are still nascent chains on the ribosome. The change in the mode of delivery disrupts outer membrane protein biogenesis, but the molecular mechanism is unclear. We sought to understand the molecular basis for the disruption of outer membrane biogensis using genetics. We partially acheived this goal by isolated a large number of mutants that are more resistant to SDS/EDTA treatment. We also explored the ability of protease inhibitor proteins to suppress the increased sensitivity of TF mutants to SDS/EDTA. Our preliminary results suggest that cells lacking TF are sensitised to blocking of the membrane-embedded Sec machinery (SecYEG) by arrested ribosomes, which results in degradation of SecYEG by the protease FtsH. Increased turnover of SecYEG causes a mild pleitropic defect in cell envelop biogenesis. However, the technician who was doing this work had to take leave of absence and is just now finishing this work. |
| Exploitation Route | Our work suggesting that SecA occupies three different conformations in vivo: an autoinhibited conformation, an activated conformation and a substrate-bound conformation. However, the physiological grounds for these conformations are unclear. They could ensure accurate substrate protein recognition and/or prevent non-productive interactions. One intriguing possibility is that some environmental condition results in autoinhibition of SecA (e.g. to adapt rapidly to a change in physiology). Indeed, our work suggests that treatment of cells with sodium azide (a non-physiological chemical, which mimic several physiological stresses) inhibits SecA by stabilising the autoinhibited form of SecA. One effect of azide is to disrupt binding of the C-terminal metal binding domain of SecA to iron, which perturbs its structure and stabilises the autoinhibited conformation. Amongst other topic raised by this research, future work is necessary to determine the physiological function of autoinhibition, define the structure of the autoinhibited form in greater detail and understand the physiological signicance of binding of the metal binding domain to iron. Understanding these aspects to could future researchers to exploit these findings (e.g. to improve protein production strategies, to enhance the secretion of heterologous proteins in E. coli etc). |
| Sectors | Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | Summer vacation studentship |
| Amount | £1,200 (GBP) |
| Funding ID | Zidan Yang |
| Organisation | Biochemical Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 07/2016 |
| End | 08/2016 |
| Title | A genetic screen suggests an alternative mechanism for inhibition of SecA by azide |
| Description | A high-density transposon library constructed in BW25113 was grown in the absence or presence of 0.25 mM or 0.5 mM sodium azide. In the case of the 0 mM and 0.25 mM samples, cells were grown until OD600 = 1.0. In the case of of the 0.5 mM sodium azide sample, the cells were grown to OD600 = 0.9, at which point the cells stopped growing exponentially. The location of the transposons after growth was determined using Illumina sequecing. Libraries were prepared using arbirary PCR and sequenced using single-end reads. Reads were then processed an aligned to the genome sequence for Escherichia coli K-12 strain W3110 (NCBI accession AP009048.1). Please see associated manuscript at BiorXiv for a more detailed description of the experiment. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2017 |
| Provided To Others? | Yes |
| Impact | Publication of article "Iron is a ligand of SecA-like metal binding domains in vivo" |
| URL | https://figshare.com/articles/dataset/A_genetic_screen_suggests_an_alternative_mechanism_for_inhibit... |
| Title | E. coli BW25113 azide treated vs untreated cells |
| Description | Microarray analysis of the effect of 2mM sodium azide on gene expression in Escherichia coli K-12 str. BW25113 |
| Type Of Material | Database/Collection of data |
| Year Produced | 2017 |
| Provided To Others? | Yes |
| Impact | Unclear. Data freely available through NCBI so could have unknown impact. |
| URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102322 |
| Title | Iron is a ligand of SecA-like metal-binding domains in vivo (dataset) |
| Description | EPR data accompanying the publication. Description of the data in the .DSC and data in the .DTA Bruker files. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Publication |
| URL | https://risweb.st-andrews.ac.uk:443/portal/en/datasets/iron-is-a-ligand-of-secalike-metalbinding-dom... |
| Title | Iron is a physiological ligand of SecA-like Metal Binding Domains |
| Description | NMR spectra of YecA |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Publication |
| URL | https://bmrb.io/data_library/summary/?bmrbId=27881 |
| Title | Transposon Directed Insertion-site Sequencing of a BW25113 ascA (yecA) deletion mutant |
| Description | We carried out saturated transposon mutagenesis of an ascA deletion mutant, determined the location of the transposon insertions using Illumina sequencing and mapped them to the BW25113 reference genome (CP009273.1). We have deposited three datasets: (1) the number of transposon insertions at each position in the genome (position_depth_count.txt), (2) the number of transposon insertions at each position with the transposon in orientation A (position_depth_count_pos.txt) and (3) the number of transposon insertions at each position with the transposon in orientation B (position_depth_count_neg.txt). |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Manuscript in preparation |
| URL | https://figshare.com/articles/Transposon_Directed_Insertion-site_Sequencing_of_a_BW25113_ascA_yecA_d... |