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Understanding the structural basis for carbohydrate-mediated activation of sensor proteins in bacterial two component systems

Reference	BB/D000475/1
Principal Investigator / Supervisor	Dr David Bolam
Co-Investigators / Co-Supervisors	Professor Jeremy Lakey, Dr Richard Lewis
Institution	Newcastle University
Department	Inst for Cell and Molecular Biosciences
Funding type	Research
Value (£)	214,527
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	30/09/2009
Duration	46 months

Abstract

Two component signalling pathways, comprising a sensor protein and response regulator, play a critical role in preceiving and responding to global changes (e.g. pH, temperature, redox potential and osmolarity) in their environment. Modulation of two component systems that lead to the expression of hydrolytic enzymes and the activation of secondary metabolism in industrially important prokaryotes has substantial biotechnological potential. Despite the fundamental importance of two component systems in the response of bacteria to perturbations in their environment there is a paucity of information on the precise identity of ligands that bind to the sensor proteins and how this results in the activation of these proteins. Recent studies by the applicants provide evidence that a two component system, XynS/XynR, from the saprophytic plant cell wall degrading bacterium Cellvibrio japonicus, can activate gene expression in response to the plant cell wall. The data provide the first evidence that sugar polymers, which comprise a highly diverse group of chiral biomolecules, can modulate the activity of two component systems, and also reveal that multiple ligands are able to activate a single sensor protein (XynS). Indeed, understanding how this plasticity in sugar protein recognition leads to the activation of a common signal transduction pathway will provide important new insights into how sensor proteins of two component systems are activated by different environmental cues. Furthermore, microbial-derived plant cell wall hydrolases are industrially important enzymes and thus understanding the mechanisms by which their expression is controlled will provide important insights not only into environmental gene regulation, but will facilitate the biotechnological exploitation of these hydrolytic biocatalysts. In this research proposal we will exploit a significant body of preliminary data to determine the precise ligands that activate the sensor protein XynS and how the interaction of the protein with its target carbohydrates leads to activation of the histidine kinase activity displayed by the protein. The project will make a significant contribution to our understanding of a generic prokaryotic regulatory system that plays a pivotal role in the capacity of bacteria to respond to global changes in their environment and in their synthesis of industrially important proteins. The specific objectives of the research programme are summarised as follows: 1. Elucidate, in detail, the natural ligands that bind to the sensor domain of XynS 2. Determine the crystal structure of XynS both unliganded and in complex with its target ligand 3. Use a rational design approach informed by the crystal structure of the protein to dissect the mechanism by which XynS is able to bind a wide range of carbohydrates 4. Investigate the mechanism by which the target ligands activate the histidine kinase of XynS.

Summary

Proteins on the surface of bacteria, by binding to specific molecules, sense significant changes to their external environment. The recognition of these environmental molecules activates processes within bacteria that enable them to respond to their changing environment. The presence of the plant cell wall, which consists of a plentiful nutrient, induces bacteria to produce enzymes that release sugars from the plant material, which the microorganisms can then use as an important energy source. The identification of the molecules that activate the synthesis of plant cell wall degradation, and the mechanism by which they bind to the cell surface receptor, and how this leads to the production of the hydrolytic enzymes are important industrial and biological questions. Thus, the plant cell represents the most extensive renewable industrial substrate on the planet and thus developing hydrolytic enzymes that release valuable sugars from these materials is an important biotechnological goal. The application of enzymes for environmentally-friendly recycling processes and biofuels are two of the major goals of the UK Government White Paper entitled energy for the future - renewable sources of energy. To fully exploit the industrial potential of microbes that produce these hydrolytic enzymes requires the manipulation of the signalling process that activates hydrolase production. Identification of the molecules that activate the signalling process and the mechanism by which they cause this effect will facilitate the large scale production of these industrially important enzymes. The project will also be of general biological interest. The activation of a range of microbial responses to global changes in the environment is caused by specific molecules binding to receptors on the surface of microbes. The identity of these signalling molecules and the mechanism by which they activate processes that lead to bacterial survival is poorly understood. This project seeks to provide newinsights into the identification of important environmental signalling molecules and the mechanism by which they bind to, and activate, their target receptor.

Committee	Closed Committee - Biomolecular Sciences (BMS)
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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