Chemists at Umeå University show: Structural researchers were wrong

All genetic information in higher organisms is coded in DNA and packed in the nucleus of every cell. A complex copying machinery, called transcription, ensures that the gene information is used for the structure of new proteins. Regulation of transcription is a central and vital mechanism. It enables different cell to have different purposes though all cells have the same set of genes. A large protein complex known as the mediator plays a key role in the regulation of transcription. Stefan Björklund, professor at the Department of Medical Biochemistry and Biophysics, and his research team are specialists on the function of the mediator. This protein complex consists of 25 subunits which jointly switch on or off the copying machinery of genes needed at specific times.

“The mediator acts similar to a switch in the cell. When it is out of order it causes the breakdown of transcription and possible serious diseases like cancer,” says Stefan Björklund. To understand how the mediator controls transcription, we need to know how the 25 subunits interact and collaborate.

The scientists at KBC focused in the present study on three units within the mediator: Med8, Med18, and Med20. Like all proteins they consist of amino acid chains and are only functional active when correctly folded. From his previous studies Stefan Björklund knew that these three building blocks are dependent on each other and essential for the mediator’s function. In the present study, Björklund and his research group wanted to show how the three subunits interact and bind to each other.

Luckily Stefan Björklund just found the perfect collaboration partner at KBC. Professor Pernilla Wittung-Stafshede and her team at the Department of Chemistry study the mechanisms of correct protein folding. As Björklund and Wittung-Stafshede both live on the same street in Berghem, Umeå, they could also discuss their research during their free time. They came very quickly to a completely unexpected solution of a problem that German researchers left behind after solving parts of the structure of the Mediator complex.

Graduate student Zaki Shaikhibraihim and post-doc Hamidur Rahaman performed the studies on Med8, Med18 and Med20 from baker's yeast cells, which use the same regulation mechanism of transcription as human cells do.

“First, we studied the three building blocks separately and how they fold to active proteins,” says Pernilla Wittung-Stafshede. Then, we combined two of the three proteins at a time. We found that Med8 could fold and bind a pair with Med18 and Med18 could do the same with Med20.

But the unexpected happened when the scientists tried to add the third subunit to the Med8-Med18 and Med18-Med20 pairs. Surprisingly, the folded protein pairs were not able to bind with the third protein. Moreover, the protein pairs Med8-Med18 and Med18-Med20 seemed to be misfolded and had lost their function. Only, when the three subunits were combined in an unfolded state, the research team managed to get all three proteins to form a functional active and correctly folded protein complex, a “trimer”.

Stefan Björklund, who led the study, is convinced that the new results are important for future research of large protein complexes. The previously published structure of the protein pair Med18-Med20 could not have been accurately folded because it was not able to pair with the third protein Med8.

“Since collaboration at the Chemical-Biological Centre spans across faculty and departmental borders, we could prove the earlier findings of German scientists with new methods and got new and exciting results,” says a proud Stefan Björklund.

Umeå researchers' results show that it is not only sufficient to describe the crystal structures of individual proteins or even parts of larger complexes. Pernilla Wittung-Stafshede and Stefan Björklund agree that studies of biologically relevant structures of large protein complexes (such as mediator) must also consider the dynamics and timescales of binding and folding reactions. The new findings from the Umeå chemists also show that the binding of protein units to each other before folding may be a more important pathway than previously thought for the formation of large protein complex.

Contact: Professor Stefan Björklund, phone: +46 (0)90 786 67 88
e-mail: stefan.bjorklund@medchem.umu.se

Reference:
Z. Shaikhibrahim, H.Rahaman, P. Wittung-Stafshede, S. Björklund: Med8, Med18, and Med20 subunits of the Mediator head domain are interdependent upon each other for folding and complex formation. Proceedings of the National Academy of Sciences of the United States of America (PNAS),
Early online edition: doi 10.1073/pnas0907645106.