Assembly and Dynamics of Bacterial Chemosensory Signaling Arrays

For nearly six decades, chemotaxis - a ubiquitous biological behavior enabling the movement of a cell or organism toward or away from chemicals -has severed as a paradigmatic model for the study of cellular sensory signal transduction and motile behavior. The relatively simple chemotaxis machinery of the bacterium Escherichia coli is the best understood biological signal transduction system and serves as a powerful tool for investigating the molecular mechanisms that proteins use to detect, process, and transmit stimulus information. E. coli cells respond to changes in their chemical environment through a sensory apparatus that is an ordered array (chemosensory array) of hundreds of basic core signalling units consisting of three essential components, the transmembrane chemoreceptors that detects the environment, the histidine kinase that passes the signal to the downstream effector, 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. In bacterial pathogens, chemotaxis response is crucial for colonization and infection. Thus, the signal transduction systems that mediate such responses are potential new targets for antimicrobial drug development.

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, and its dynamical properties. In this project, we propose to use a combination of cutting-edge cryoEM structural methods and computational modeling and multi-scale molecular simulations, as well as in vivo functional assays for structural validation, to investigate the structural and dynamical mechanisms underlying signal transduction and regulation in the chemosensory array. Our results will establish, in atomistic detail, how individual signals are transmitted across the receptor, adaptively regulated, and subsequently integrated over multiple receptor proteins to jointly affect kinase activity, highlighting general features of cooperative protein signaling. The significant overlap in molecular machinery employed by diverse chemotactic species will greatly extend the relevance of our results, including to signal transduction within a wide-range of human and plant pathogens.

The mechanism of stimulus-response coupling in bacterial chemotaxis has emerged as a paradigm for understanding the principles of intracellular signal transduction both in bacterial and eukaryotic cells. Bacteria use chemotaxis signaling pathways to monitor and response their environment changes. The essential core signaling unit comprises transmembrane receptors, a histidine kinase CheA, and a coupling protein CheW. A few hundred core signaling complexes assemble into a lattice array responsible for the remarkable cooperativity in chemotaxis signaling. Despite current atomic structures of the individual soluble domains of the receptor, CheA, and CheW and sub-complexes of the core unit, high resolution structures of the full core complex and its extended higher order array assembly, as well as the conformational states associated with signal transduction and transmission, have remained unattainable. The large size and dynamic nature of the signalling array have challenged conventional structural methods. We recently developed a novel in vitro reconstitution system to generate arrays of the core signaling units that mimic the native chemosensory apparatus, and have obtained a density map of the array at 9Å resolution by cryo-electron tomography (cryoET) and sub-tomogram averaging of data recorded on a CCD camera. Building on this success, we aim to 1) determine the structures of the core signaling complex and its extended array to near-atomic resolution using new direct electron detector and cryoET and sub-tomogram averaging, 2) develop atomic models of the dynamic signaling arrays by integrating the cryoEM structural "snapshots" of different states with large-scale MD simulations, and 3) functionally characterize the molecular interfaces within native cells. Through this integrative approach, the proposed studies will provide new and comprehensive insights into the mechanisms of chemotactic array assembly, activation and cooperativity.

Understanding the mechanism of signaling in bacterial chemotaxis is of great interest for many areas of biology and medicine. Our proposed efforts for a comprehensive and integrated structural and functional analysis of the conserved bacterial chemosensory array will provide new insights into receptor-kinase coupling, signaling complex and array formation, and conformational dynamics essential for signaling, and will contribute to a deep understanding of signal transduction and signal processing that will also present new targets for antimicrobial drug development. Impact will arise in several ways.

Firstly, our work will inform fundamental microbiology, providing structural paradigms that explain function.

Secondly, it will provide new methods, protocols, reagents and software tools that may be of general value to structural biologists and computational biologists.

Thirdly, the proposed study is basic research of potential value to the commercial sector, in particular in the biotechnology and pharmaceutical industries on antimicrobial drug development. By inhibiting the proteins involved in chemotaxis, one can develop novel antibiotics to control multi-drug-resistant strains of bacteria. Given, the drug discovery process can take up to 20 years, it is imperative we prepare well in advance to counteract the threat of a society where resistant bacterial strains are common-place and untreatable. Therefore, if we are sufficiently well equipped, the impact of this proposal will be long lasting and life-changing for the future generations.

Fourthly, increased public understanding is an important benefit to the wider public. Structural and Computational approaches to the biosciences has a major advantage in that they are able to produce artistic and descriptive illustrations of biomolecules, that facilitate the accessibility of these ubiquitous macromolecular machines to the general public. Furthermore, molecular simulation enables the reanimation of statically resolved structures into movies that demonstrate the dynamics visually. By tuning the science to an appropriate level of detail, by using graphics tools such as Blender, one can make the research available as museum displays and as educational tools in schools.

The final area of impact for this research project will be in the training and career development of a cohort of young research scientists. We have been able to work with very talented, energetic, enthusiastic scientists, some experienced, and others at an early career stage, and we believe our laboratories have provided them with an excellent training environment. Of the order of a dozen of these have since gone on to leadership positions, in academia and industry.

This work is directly related to the BBSRC strategic priority areas of Bioscience for Health ("Develop and apply new tools in areas such as chemical biology, high resolution structural analysis"), to World-class Bioscience ("predictive, integrative and systems approaches in bioscience at a range of scales from molecules to...") and to Exploiting New Ways of Working by developing "the next generation of bioscience tools to drive new and deeper understanding in bioscience". This proposal directly relates to Combatting antimicrobial resistance by studying digital pathways within pathogenic organisms, while there are also elements to this proposal that comprise the systems approaches to and technology development for the biosciences. Therefore, this promotes partnerships with the pharmaceutical industry, in addition to the development of academic collaborations.

Thus, the overall impact will be to advance UK knowledge and technological development as well as promoting health and wellbeing through better drug design. Ultimately this will add significantly to the competitiveness of UK industry.