Study reveals the subtleties of molecular recognition in bacteria
Professor Judith Armitage and her group in the Biochemistry Department, together with other researchers in the University, have made the first steps towards being able to identify interacting partners in signalling pathways and potentially engineer a bacterial cell that can sense and respond to novel environmental cues.
The research groups of Professor Armitage, Director of the BBSRC-funded Oxford Centre for Integrative Systems Biology in the Department, and Professor David Stuart in the Division of Structural Biology at the Wellcome Trust Centre for Human Genetics, have just published their work in PloS Biology.1
Interacting partners in a signalling pathway. Structure of a sensor-response regulator complex from Rhodobacter with a portion of the sensor protein shown in blue and the response regulator in green. The background image shows Rhodobacter with the intracellular protein cluster that transmits the external signal tagged in green
Being a single-celled organism, a bacterium must be able to react to whatever insults the environment throws at it. Changes in the environment are relayed through the bacterial cell via signalling mechanisms.
One widely-used mechanism is a two-component signalling circuit made up of a type of protein called a kinase, which acts as the sensor, and its partner protein known as a response regulator. A single cell can encode over 100 related but distinct two-component pairs.
Chemotaxis, an important process which controls the movement of the bacterium when it senses a chemical or nutrient gradient around it, is regulated by one of these two-component signalling pathways.
Professor Armitage's group works on the chemotaxis system in the bacterium Rhodobacter sphaeroides. Rhodobacter uses multiple highly homologous sensor-response regulator pairs to relay external cues to the motor that drives movement of the bacterium. How the specificity of these pairs is tightly controlled to prevent 'crossed wires' between signalling pathways has been unclear until now.
"The response regulators all look very similar in terms of their structure," explains Christian Bell, the postgraduate student who worked on the project in both labs. "So what we aimed to find out was how one sensor protein can distinguish between many response regulators."
The two Oxford groups combined their expertise in biochemistry and structural biology to tackle the problem. They used extremely bright pinpoints of light produced by Diamond Light Source, the synchrotron facility in Oxfordshire, to carry out X-ray crystallography. This method allows visualization of proteins at an atomic level.
Using this approach, the researchers solved the three-dimensional structure of one of the two-component complexes in Rhodobacter. They were then able to pinpoint the specific amino acids that are required for this molecular recognition.
"What was surprising was that it is really just one or two amino acids that drive the interaction between the proteins, " explains Christian. They found that one amino acid on the response regulator pointed out like a finger towards a pocket on the sensor, enabling the two proteins to fit snugly together.
Having identified these key amino acids, they attempted to re-engineer the chemotaxis circuitry by introducing this finger into other response regulator proteins which do not normally partner this specific sensor.
Structure of the sensor-response regulator complex from a different view with the critical 'finger' shown in stick representation
"We took the other four chemotaxis response regulators in Rhodobacter and introduced the key amino acids and found that they all bound to the sensor," says Christian. Once bound, the sensor chemically modified the response regulators as it would its native partner.
This is the first time that researchers have re-designed the intracellular part of the chemotaxis circuitry, paving the way for producing custom-designed circuits for applications in synthetic biology. The work should also help in the development of tools to identify interacting pairs of sensory proteins from genome sequences.
"This is a significant step along the road to rational design of protein signalling networks," says Professor Armitage, commenting on the implications of the work. She also adds that the work shows the strength of collaborations between biochemists and structural biologists.
The potential of this type of work in the future is exciting, as Christian explains. "The aim is to understand the system so well that you're able to change it in any way you like. The dream will be a synthetic cell that does exactly what you want."