Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS


MEMBRANE PROTEINS Informe resumido

Project ID: 9929
Financiado con arreglo a: FP6-MOBILITY
País: France

Final Activity Report Summary - MEMBRANE PROTEINS (Structural characterisation of the type III secretion translocon of Pseudomonas aeruginosa)

Certain bacteria are accomplished saboteurs, attacking human cells with a sophisticated arsenal evolved to overcome the cell's protective barrier, such as skin or mucosa. The type III secretion system (T3SS) is a particularly dangerous weapon. It is employed by pathogens such as yersinia pestis, or bubonic plague, Escherichia coli, i.e. diarrhoea, and pseudomonas aeruginosa, which causes opportunistic infections in persons with weakened immune systems, such as acquired immune deficiency syndrome (AIDS), cancer or burn patients, causing great suffering and thousands of deaths annually.

The T3SS is a sturdy protrusion sitting on the bacterial surface. It acts like a molecular syringe, injecting toxic molecules straight into the human cell. For this to be effective, the tip of the syringe needle has to connect with the inside of the human cell. In other words, the needle has to poke through the cell membrane. The bacterium accomplishes this by producing PopB, a protein that is secreted through the needle and subsequently inserted into the human cell membrane. In there, PopB spontaneously assembles into a pore-like complex that the needle connects to, creating a continuous passage from the bacterium into the human cell, a passage through which the bacterial toxins can now travel.

PopB is a peculiar protein. Once produced in the pathogenic bacterium, they embark on a long journey that includes squeezing through the interior of the syringe needle and inserting into a membrane. Proteins normally do not do that sort of tricks. They are either in the cell interior or in the membrane. They are either happily folded or squeezed out of shape. PopB does not fit into these conventions. It can change its shape and environment easily. This makes it a very interesting protein to work with, but it also makes it very challenging.

We set out to understand the mature pore. At first, we studied how many PopB molecules were needed to come together and form the pore. This could not be directly done since the pore was unstable in a test tube. An experiment providing striking results was chemical cross-linking. In this case, a chemical with two identical reactive groups was added to the solution containing pores. The reactive groups could interact with two protein molecules simultaneously in case those were in close proximity, which we expected if they existed as pores. Simply said, the pore was locked up by chemical glue. The size of the pore could now be determined and, from the known size of a PopB molecule, the number of molecules per pore could be also defined. It turned out that six PopB molecules formed a pore. This result was also confirmed by other means.

At a second stage, we wanted to determine the exact molecular structure of the pore. The gold standard for structural work was crystallisation, followed by X-ray irradiation. A protein crystal diffracted X-rays much like narrow slits diffracted visible light. From the pattern of diffraction one could calculate the position of the atoms in the molecule which, when linked up, gave the structure.

Despite tremendous progress in the field, crystallising a protein was still a formidable challenge. Whereas salt could be crystallised from a solution simply through water evaporation, proteins crystallised under a few specific conditions that had to be determined anew for each protein. These conditions were yet fewer and harder to find for proteins like PopB that normally resided in membranes.

Against these odds, initial crystals were obtained towards the end of the project. These were still too small to be useful in diffraction experiments but served to set the pace of future research. In an atmosphere of optimism, a spirited student joined the laboratory to prepare his doctoral thesis on the structure of PopB.


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