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

Completed

Not Energy Related
Renewable Energy Sources(Bio-Energy, Other bio-energy)

Basic and strategic applied research

BIOLOGICAL AND AGRICULTURAL SCIENCES (Biological Sciences)

Not Cross-cutting

Dr D Bolam
Institute for Cell and Molecular Biosciences (ICaMB)
Newcastle University

Research Grant

BBSRC

01 December 2005

30 September 2009

46 months

£214,527

Transport Systems and Vehicles

North East

Dr D Bolam, Institute for Cell and Molecular Biosciences (ICaMB), Newcastle University

Prof J Lakey, Institute for Cell and Molecular Biosciences (ICaMB), Newcastle University
Prof R Lewis, Institute for Cell and Molecular Biosciences (ICaMB), Newcastle University

Objectives not supplied

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.

22/11/13