Award details

The mechanism of SecA-dependent substrate recognition and delivery in Escherichia coli

Reference	BB/L019434/1
Principal Investigator / Supervisor	Dr Damon Huber
Co-Investigators / Co-Supervisors
Institution	University of Birmingham
Department	Sch of Biosciences
Funding type	Research
Value (£)	397,618
Status	Completed
Type	Research Grant
Start date	29/09/2014
End date	31/12/2017
Duration	39 months

Abstract

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.

Summary

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 theCM, 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.

Impact Summary

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.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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