Dynamic allostery of Sec machinery in protein transport and folding

All cells are surrounded by oily membranes keeping the cell insides in, and unwanted substances and parasites out. Thus, selective pores have evolved for the supply of nutrients and removal of waste products. Without these 'transporters' the membrane presents an impermeable barrier, particularly for larger molecules like proteins. Such passage is required for many proteins to perform their biological functions, such as secretion of antibodies into the blood stream by immune cells. Therefore, biological membranes also contain a number of translocation systems that recognise the specific proteins to be translocated via signals embedded in the sequence of composite amino acids.
We aim to learn more about how one such translocation system works in bacterium Escherichia coli: the "translocon", which secretes proteins from the interior across the membrane and also into it; essential even for basic survival. The machinery comprises two components -a protein-channel embedded within the membrane, and a motor protein named SecA that drives the passage of proteins through the channel, fuelled by energy provided by ATP, the so-called "energy-currency" of the cell.
The energy for protein translocation is released when SecA breaks down ATP into two smaller molecules, ADP and phosphate and this process is coupled to the movement of the translocating protein in a cycle of changes to the shape of SecA and the channel, termed conformational changes. Since there are two partners, we are particularly interested how their movement is coordinated. Our objective is to understand how this "dance" is choreographed in time and space, i.e. take a molecular movie of the pair.
We have developed tools, which allows us to interrogate one component at a time with a high temporal resolution by illuminating one of the partners at a time. This is achieved with rapidly alternating laser pulses, while recording their behaviour by aiming very sensitive and fast detectors (or cameras) onto the dance floor. To do so, we have to use genetic and biochemical techniques to introduce optical reporters (fluorescence probes) onto the proteins, in places that move during the translocation process, or dance (if you prefer our colourful metaphor!).
These fluorescent probes report on the specific motions during different stages of protein translocation and during ATP breakdown. In the past few years, we have had considerable success in this approach observing molecular gyrations of individual partners. Surprisingly, we found the translocon dances about ten times faster than its energy providing motor partner. Now, we want to figure out how this is possible, and how such a strange dance is coordinated. To do so we will observe movies of both partners simultaneously, which requires considerable molecular biology craft for the introduction of the right reporters in the right places. We also need more elaborate and faster laser systems to illuminate the dance floor and of course fast rolling cameras and other detectors. After the movie is recorded, we will tease out useful information about the dance from individual movies that are encoded in the detected signals. To do this we have developed computational algorithms to convert the extremely rapid signals into meaningful information.
The findings will uncover details of the conformational dance and explain fundamentally how the coordinated, and paradoxically incoherent, behaviour of the motor and channel result in the passage of proteins across and into the membranes of cells. This information could also inform us about how very different dancers move elsewhere in biology, even with different routines. So, we could learn more how ATP is also used to, help move different types of cargo around the cell, and for protein quality control. Moreover, this knowledge could be exploited in the development of compounds that target those elements (dancers!) that are unique to bacteria, potentially for highly desirable novel antibiotic.

The proposal aims to delineate the molecular mechanism of protein translocation by the Sec system. This machinery provides the main pathway for protein secretion and membrane protein insertion across cellular membranes, and is conserved among all forms of life. Transport of proteins across the bacterial inner membrane occurs primarily at the SecYEG translocon. In this case, secretion mostly occurs post-translationally, with the help of cytosolic ATPase SecA, which associates to drive the protein through the SecY-channel, using energy from ATP hydrolysis and the trans-membrane proton motive force (PMF). Structural biology has revealed the arrangements and interactions between SecYEG and SecA, and provides framework for the project. However, despite this detailed information, the central question of how a polypeptide is dynamically translocated through, or into, the membrane remains to be answered.
This proposal builds on our discovery of two-way allosteric communication between the SecA ATP binding site and the central channel of SecY enabled by advances in single molecule detection. We aim to elucidate the mechanism of this dynamic allosteric coupling and the role of pore dynamics during protein translocation for: secretion, as well as for integral membrane protein insertion and outer membrane protein folding and insertion. We will use a combination of biochemistry and time-resolved single molecule fluorescence and computational tools to follow translocation, and corresponding conformational changes. Förster resonance energy transfer (FRET) will allow real time dynamic reporting for interpretation in the context of the available high-resolution structures.
The results will address the key outstanding question: how rapid, stochastic gating of the translocon is allosterically coupled to slower ATP hydrolysis at the SecA motor, and whether such dynamic coupling is required to fulfil additional insertion and downstream functions of this versatile membrane machinery.

The overarching aim of the proposal is to gain an understanding of an important fundamental aspects of bacterial biology: protein secretion, membrane protein insertion and Gram-negative outer-membrane biogenesis. The immediate impact in terms of the current project will lie in scientific advancement and the generation of new knowledge. The project will also present new hypothetical concepts that if proven to be true will have a major impact in our understanding of protein transport, and have important implication for the development of effective treatments against bacterial infections. An additional goal is to encourage a broader uptake of the technological applications we are helping to develop for exploitation in fundamental studies in both academic and commercial sectors.
The main areas of impact are:
1. Application and exploitation. While the proposed project is at a "pre-competitive" stage in terms of commercial exploitation, the knowledge generated will have an immediate benefit to both the National and International bioscience community (academic and commercial) in terms of understanding a fundamental process that spans the breadth of biology. The process is of fundamental importance for bacterial survival and certain complex components are specific to bacteria. The bacterial envelope and its biogenesis are particularly vulnerable to attack; its weakening by, for instance, antibiotics can be lethal. Therefore, the subject of this proposal is a particularly fertile area, with respect to the development of new antibiotics and for strategies against anti-microbial resistance (AMR). Therefore, in the medium term the work could lead to new approaches/ targets for antimicrobial drug development. The knowledge gained could support an ongoing work aimed towards a drug discovery programme in Bristol.
2. Development of new technology. Single molecule detection is emerging as important screening tool as demonstrated in Leeds by the development of sensitive methods to follow virus assembly and screen for anti-virals.
Both Leeds and Bristol have mechanisms in place to increase the impact of research and to exploit any commercialization.
3. Engagement. The benefits to the bioscience community are summarised above. The standard routes to information dissemination (e.g. pre-print submissions, papers in journals and presentations at conferences) will be used throughout the duration of the project. A more general benefit of our work to the UK stems from our commitment to public engagement. The PI and PDRAs routinely participate in public engagement activities, from school children to politicians, and for the promotion science to women and girls. The group will continue with public engagement activities throughout the course of the project, using work generated from the project to exemplify the importance of research.
The PIs also interact with pre-university students with the aim to excite them about the research process in order to encourage them to pursue a future in the high value field of research and development. The PIs will continue with these activities throughout the course of the project, using work generated from the project to exemplify the importance of research.
4. The critical collaboration proposed with the Tuma group in the Czech Republic, which will enhance the value of research in the UK and maintain the UK's scientific European research network, which post-BREXIT will be more important to maintain than ever before.
5. Staff training. The project will generate trained staff with desirable expertise in complex biochemical and biophysical analysis of membrane protein complexes that are involved in important bacterial activities. The researchers will be in demand in both the academic and commercial sectors. During the project, further development will be encouraged through attending courses in areas directly and indirectly related to their role as research scientists (e.g. management and leadership).