Periodic Reporting for period 2 - ChemoTaxi (Molecular choreography of bacterial chemotaxis signalling)
Reporting period: 2023-03-01 to 2024-08-31
For nearly six decades, chemotaxis - a ubiquitous biological behaviour enabling the movement of a cell or organism toward or away from chemicals -has served as a paradigmatic model for the study of cellular sensory signal transduction and motile behaviour. The relatively simple chemotaxis machinery of E. coli is the best understood signal transduction system and serves as a powerful tool for investigating the molecular mechanisms that proteins use to detect, process, and transmit signals. The sensory apparatus of E. coli cells is an ordered array of hundreds of basic core signalling units consisting of three essential components, the transmembrane chemoreceptors, the histidine kinase, and the adaptor protein. The core units further assemble into a two-dimensional lattice array which allows cells to amplify and integrate many varied and possibly conflicting signals to locate optimal growing conditions.
To understand the underlying molecular mechanisms of chemosensory array assembly, activation and high cooperativity, it is essential to determine the precise interactions between the core signalling components in the context of the array. We propose to use a combination of cutting-edge cryoET structural methods and multi-scale molecular simulations, as well as in vivo functional assays, to investigate the structural and dynamical mechanisms underlying signal transduction and regulation. The research plan is divided into three aims:
1. Determine the structural basis of signal transduction and array cooperativity
2. Define conformational states and dynamics of the array
3. Obtain time-resolved structural snapshots of signalling pathway
Our results will establish, in atomistic detail, the chemotaxis signalling pathway that is shared by diverse chemotactic species, including a wide-range of human and plant pathogens, and thus impact on multiple disciplines, from antimicrobial drug development to understanding responses to hormones and neurotransmitters in eukaryotic cells.
To understand the underlying molecular mechanisms of chemosensory array assembly, activation and high cooperativity, it is essential to determine the precise interactions between the core signalling components in the context of the array. We propose to use a combination of cutting-edge cryoET structural methods and multi-scale molecular simulations, as well as in vivo functional assays, to investigate the structural and dynamical mechanisms underlying signal transduction and regulation. The research plan is divided into three aims:
1. Determine the structural basis of signal transduction and array cooperativity
2. Define conformational states and dynamics of the array
3. Obtain time-resolved structural snapshots of signalling pathway
Our results will establish, in atomistic detail, the chemotaxis signalling pathway that is shared by diverse chemotactic species, including a wide-range of human and plant pathogens, and thus impact on multiple disciplines, from antimicrobial drug development to understanding responses to hormones and neurotransmitters in eukaryotic cells.
We have succeeded in progressing our structural studies on the complete native chemosensory core signaling unit (CSU) from the phage E-lysed E. coli cells. The structure was determined using cryo-electron tomography (cryoET) and sub-tomogram averaging (STA), from which we built the atomic models of the CSU’s constituent proteins as well as key protein-protein interfaces, enabling the assembly of an all-atom CSU model. Molecular dynamics simulations of the resulting model provide new insight into the periplasmic organization of the complex and interactions between the neighbouring CSUs in the array. Our results further elucidate previously unresolved interactions between individual CheA domains, enhancing our understanding of the structural mechanisms underlying CheA signaling and regulation. The manuscript is published in mBio last year.
We have also made excellent progress in the structural analysis of CSUs from monolayer arrays. One main challenge in this in vitro reconstituted system is the preferred orientation problem. Using the latest cryoEM technology, specifically access to a developmental Krios equipped with a 90-degree tilt stage, we devised a novel data collection strategy to overcome the preferred orientation problem and achieved better than 7 Å resolution. The manuscript is being prepared for publication.
During this first period of ChemoTaxi we set out to build up expertise in the team to apply genetic alteration, molecular biology, and cutting-edge cryoET methods to dissect the conformational states of the chemosensory array. We have generated many mutants of CSUs both for in vitro monolayer arrays and for in vivo native arrays with an E-lysis system. These mutants are being characterized, which will also be incorporated in Aim 3 in E. coli minicells for time-resolved studies. In parallel, we were successfully awarded Archer2 time (28,800 CUs) via HECBioSim, and we have also set up the simulation systems in Baskerville HPC (free access for Diamond).
For time-resolved structural snapshots of the chemotaxis signalling pathway, we have carried out two processes in parallel: i) We have obtained caged-serine and have now characterized the photophysical and biochemical properties of the caged compound and optimized the parameters and conditions for time-resolved signalling on cryoEM grids. ii) We are setting up a new method, laser-controlled devitrification and revitrification of cryoEM samples, for time-resolved structural studies. This is in collaboration with Prof. Angus Kirkland at the Rosalind Franklin Institute. The method is based on a very exciting recent work by Lorenz’s group in EPFL (Harder et. al, 2023 Nat Commun).
Lastly, since the start of this project, we have developed several novel cryoEM methods and technologies to advance in situ structural biology, including three software packages, emClarity, IceBreaker, and MagpiEM.
We have also made excellent progress in the structural analysis of CSUs from monolayer arrays. One main challenge in this in vitro reconstituted system is the preferred orientation problem. Using the latest cryoEM technology, specifically access to a developmental Krios equipped with a 90-degree tilt stage, we devised a novel data collection strategy to overcome the preferred orientation problem and achieved better than 7 Å resolution. The manuscript is being prepared for publication.
During this first period of ChemoTaxi we set out to build up expertise in the team to apply genetic alteration, molecular biology, and cutting-edge cryoET methods to dissect the conformational states of the chemosensory array. We have generated many mutants of CSUs both for in vitro monolayer arrays and for in vivo native arrays with an E-lysis system. These mutants are being characterized, which will also be incorporated in Aim 3 in E. coli minicells for time-resolved studies. In parallel, we were successfully awarded Archer2 time (28,800 CUs) via HECBioSim, and we have also set up the simulation systems in Baskerville HPC (free access for Diamond).
For time-resolved structural snapshots of the chemotaxis signalling pathway, we have carried out two processes in parallel: i) We have obtained caged-serine and have now characterized the photophysical and biochemical properties of the caged compound and optimized the parameters and conditions for time-resolved signalling on cryoEM grids. ii) We are setting up a new method, laser-controlled devitrification and revitrification of cryoEM samples, for time-resolved structural studies. This is in collaboration with Prof. Angus Kirkland at the Rosalind Franklin Institute. The method is based on a very exciting recent work by Lorenz’s group in EPFL (Harder et. al, 2023 Nat Commun).
Lastly, since the start of this project, we have developed several novel cryoEM methods and technologies to advance in situ structural biology, including three software packages, emClarity, IceBreaker, and MagpiEM.
By developing advanced methods for in situ structural biology (software and hardware), we decipher structures of native nucleosomes and show how individual nucleosomes are organized into chromatin fibres within the intact frozen-hydrated T-lymphoblast CEM cell nucleus using cryo-focused ion beam (cryo-FIB) and cryo-electron tomography (cryo-ET) and subtomogram averaging. The revelation of the native chromatin fibre structure in the intact cell nucleus in situ was an unexpected breakthrough, advancing the research field significantly beyond the state of the art. Chromatin plays pivotal roles in life processes in eukaryotes, and its structure lays the very foundation for its function. While studies from in vitro assemblies suggest competing models for the 30-nm chromatin fibre, the existence of the 30-nm chromatin fibre in the native cells has been debated over the past decades and the structure of native chromatin fibres remains elusive to date. This work not only rewrites the textbook on chromatin organization but also opens a new avenue for high-resolution in situ investigations of gene expression, replication, repair, and many DNA-associated processes.
The development of these advanced technologies will enable high-resolution structures of chemotaxis signalling arrays within the native bacterial cells and in multiple conformation states, for the understanding the underlying molecular mechanisms of chemosensory array assembly, activation, and high cooperativity.
The development of these advanced technologies will enable high-resolution structures of chemotaxis signalling arrays within the native bacterial cells and in multiple conformation states, for the understanding the underlying molecular mechanisms of chemosensory array assembly, activation, and high cooperativity.