Desiphering the structural origins of functional multimodality in bacterial mechanosensitive ion channels

Ion channels are central to life sciences due to direct involvement in signal transduction, aging, cancer and neurodegeneration. Despite progress in the fundamental understanding of the structure and function of specific membrane proteins (Nobel Prizes in Chemistry 1988, 1997, 2003 and 2012), this has only been the tip of the iceberg. There is now an immediate need for the development of novel triggers to control pores in the cell membrane of deadly pathogens and tackle antimicrobial resistance, the most common cause of death worldwide.

We anticipate to achieve that by exploiting an ancient and ubiquitous mechanism of ion channel regulation named mechanosensation. The latter is the ability of membrane proteins to sense tension changes occurring within the lipid membrane and respond to these by altering their structure and function. This proposal aims to gain a fundamental understanding of mechanosensation and decipher the molecular basis of its co-existence with other forms of ion channel regulation.

To this end, we will dissect the individual steps that form its molecular basis and identify the common, but essential structural elements responsible to transduce pressure-sensing abilities to channels. The fundamental aspect of the underlying impact of the lipid membrane in ion channel regulation along with the physiological role of the functional versatility of pressure sensitive channels will be elucidated. To this end, we will identify the unique structural features which allow functional multimodality of mechanosensitive channels and their ability to respond to other stimuli, such as ions, pH or specific molecules, in addition to mechanical triggers.

Within the project we will develop and follow an integrated multidisciplinary approach in order to establish a link between mechanical-activation and ligand-gating. The proposed studies will involve using a suite of state-of-the-art structural (Cryo Electron Microscopy and X -Ray crystallography), biochemical (Protein Purification), biophysical (Electron Paramagnetic Resonance spectroscopy and Electrophysiology) and sophisticated computational methods (Molecular Dynamics) to address questions pertaining to the mechanism and regulation of distinct members of the bacterial mechanosensitive ion channel family at a molecular level.

Collectively, we anticipate to translate forces within the membrane participating in the mechanical activation of channels into specific molecular stimuli, which would mimic mechanotransduction. Similar to optogenetics, a field which has massively evolved over the last years and is based on the interaction of proteins with the ancient physical property of light, pressure sensing, one of nature's most fundamental regulatory mechanisms, would enable a radically novel field to emerge.

We aim to better understand ion transport across cell membranes in pathogenic bacteria - the most common cause of death worldwide - via mechanistic and structural studies of a class of integral membrane proteins called mechanosensitive (MS) ion channels, ubiquitous across life kingdoms. An arsenal of advanced structural, biochemical and biophysical methods will be employed to elucidate the molecular basis of the underlying property which regulates these systems, that is mechanical sensing. Importantly, we have already developed a pipeline for production of pure, functional, the MS channel proteins MscK, MscM and YbiO to underpin the work. Functional characterisation will utilise purified wild type channels reconstituted into artificial lipid membranes called liposomes that mimic the cellular environment to allow for a detailed study of ion kinetics and mechanism by exploiting electrophysiology. These studies will also determine unambiguously the essential structural components required for mechanosensitivity of these channels, namely their ability to sense changes in tension within the lipid membrane and respond by opening their pore through which ions flow. This way these systems could convert mechanical energy into electric current. New 3D structures will be acquired by Cryo Electron Microscopy will reveal in molecular detail accurate positioning of each individual building block of the channels. This will permit to visualise crucial interactions with other proteins, ions or molecules and decipher their functional role into the regulation and physiological role of these systems in deadly pathogens. Cryo EM has revolutionarized biology after recent technological breakthroughs and the Astbury centre within the Leeds University is extremely well equipped with two state-of-the-art microscopes for solving such challenging ion channel structures. The proposed studies could further be exploited for therapeutic intervention against pathogenic bacteria.

The following groups and people will benefit from this research:
1) Academic beneficiaries: In the short term (lifetime of this grant) the main beneficiaries are academic groups, both within the University of Leeds, other UK universities and internationally. Further details are given in the academic beneficiaries section, but the approach taken in this grant proposal in terms of combining techniques to elucidate membrane channel structure (and hence function) in greater detail than previously possible, will be of great benefit to academics working on a wide range of membrane proteins.

2) Public outreach. I will strive to promote a greater public understanding of science. This will benefit the public but also the wider scientific community by highlighting the role each plays for the other. I will communicate with the public about general science but also of the specific benefits of basic biological research using model organisms. I will achieve this via outreach activities. During my time in St Andrews I have organised and actively participated through oral presentations and scientific activities to Fife College, at Stenton Campus and offered advice to local community students. In Leeds I intend to organise similar and get involved in established public outreach activities at the University of Leeds, between students in local schools of different ages and backgrounds. In particular, I will actively get involved in the Discovery Zone activities at Leeds Festival of Science for schoolchildren and the Pint of Science and the Cafe Scientifique for adults. Further, I will participate in local outreach events, such as the Great Yorkshire Show.

3) Scientific training. As part of this grant I will recruit one post-doctoral research associate (PDRA). During the course of this project, I will have to opportunity to train him/her in molecular biology, membrane protein biochemistry, crystallography and PELDOR (DEER) spectroscopy. He/She will also have access to the award-winning career development courses offered at Leeds University via a variety of organisational development and professional learning courses. I will closely mentor
the PDRA to ensure his/her career progression will be a success.

4) Societal/translational impact. Despite MS channels being present in all life forms, and that they are linked by their common property of pressure sensitivity, prokaryotic and eukaryotic MS channels are completely distinct from each other, making them excellent drug targets for either bacterial pathogens or animals/humans respectively. This presents certain advantages, such as targeting prokaryotic MscM which is present in all bacterial pathogens but absent form humans, thus making it an excellent target for tackling antimicrobial resistance. Although the work we do is at the level of fundamental basic biology, our work will help to identify MS channel targets for development of novel therapeutics. By elucidating the molecular basis of pressure sensing and its multimodal functional role within MS channels, we are taking an important first step in that direction.

5) Commercial exploitation. Despite there will be no immediate opportunities for commercial exploitation of our work, I anticipate the design of novel molecular tools for MS channel gating to create potential commercialisation opportunities. I will then take advantage to patent and/or licence any technologies resulting from our work. I have familiarised myself with the relevant authorities within St Andrews (Knowledge Transfer Centre) and I will do the same in Leeds, which has recently made a multimillion investment for industrial and commercial exploitation of patents and future spin off companies. This molecular platform will be used to identify ligands for activating or inhibiting MS channels first for in vitro studies and subsequently to animal model testing for potential biomedical applications, benefiting Leeds University and the UK's science economy.