Transport proteins are essential to all life. They are responsible for transporting molecules through the cell membrane. However, how exactly they work is still partly a mystery. Biochemists from the University of Groningen, headed by Prof. Dirk Slotboom, have resolved an important part of this mystery. On Sunday 8 September, an article by the group appeared on the site of the journal Nature Structural & Molecular Biology.
Without transportation, everything simply stops, and that also applies to every living organism. Substances have to be continually transported through the cell membrane, which forms a barrier between ‘inside’ and ‘outside’. This transport is mainly performed by transport proteins anchored in the cell membrane. These proteins bind to a specific substrate and then change in shape. This shape-change results in the substrate ending up on the other side of the cell membrane.
After this movement, the transport protein returns to its starting position. However, there is a problem that has kept scientists busy for years, explains Associate Professor of Biochemistry Dirk Slotboom of the Groningen Biomolecular Sciences and Biotechnology Institute of the University of Groningen. ‘After the return, what fills the place where the substrate used to be? It’s simply not possible to have an empty space in a protein.’
He investigated this in the protein complex GltPh, which is responsible for absorbing the nutrient aspartate in microorganisms. The mechanism used by GltPh to transport aspartate from inside to outside can be compared to a lift – a door opens outside and the aspartate nips in to fill a space in the protein. The door then shuts and moves part of the protein from outside to inside, taking the aspartate with it. Once inside, another door opens and the aspartate moves out of the space into the cell. The door shuts again and the lift returns to its starting position.
In order to see what was in the empty lift, the scientists had to create crystals from the empty protein. These were then analysed with X-rays. The scattering of X-rays enables the three-dimensional structure of the protein to be calculated. Although X-ray analysis of protein crystals is a common technique to acquire this sort of information, the crystallization of empty transport proteins is very tricky. ‘This is because these proteins are in the cell membranes, and are not easy to pack them in a crystal lattice, explains Stotboom. PhD student Sonja Jensen was able to find the right biochemical conditions for the crystallization. Together with postdoc Albert Guskov, she then determined the three-dimensional structure of the empty protein.
The analysis revealed that the protein used one of its own amino acids to fill the empty space. Proteins consist of long chains of 20 different amino acids that are folded into a complex pattern. The aspartate transporter GltPh is 450 amino acids long. An arginine amino acid is at position 397 in this protein. Part of this arginine residue turns out to fill the space left by the aspartate in the empty lift.
This is the first time that a research group has shown what fills the empty space in a transport protein like this one. This throws new light on confusing results from studies on similar transport proteins in the brains of humans and rats. In our brains, these transport proteins are responsible for glutamate, a signalling molecule used by nerve cells to communicate with each other, remaining stored in cells until a nerve impulse arrives. Mutagenesis of the corresponding arginine cripples these transport proteins in the brain – the lift can still move in the presence of glutamate but an empty lift cannot return.
The link to the proteins in the brain makes this research medically significant. Nerve cells that use the signalling molecule glutamate in the brain are involved in the memory and the ability to learn. When these proteins do not work properly, diseases such as Alzheimer’s, ALS or Huntington’s can occur. These transport proteins also play a role after damage to the brain as the result of a stroke. By better understanding how these proteins work, it may be possible in the long term to intervene in the event of these diseases or brain damage.
For Dirk Slotboom, the research is a step in a longer process, in which understanding how the transport proteins function is what he is interested in. This will need more than static images of the proteins, produced with the help of crystallography – dynamic information is needed. Working with the group run by his Groningen colleague Prof. Antoine van Oijen, he is now trying to chart the movements in the lift.
Contact details: Prof. Dirk Slotboom
Crystal structure of a substrate-free aspartate transporter
Sonja Jensen, Albert Guskov, Stephan Rempel, Inga Hänelt, and Dirk Jan Slotboom
Nature Structural & Molecular Biology, doi:10.1038/nsmb.2663
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