The Mystery of the Empty Lift

Forget trucks, trains or cargo ships: the most vital form of transport is in our bodies. Our cells contain molecules or complexes that transport important substances in and out of them. But despite their importance, these transporters are still veiled in mystery. Biochemists and physicists from the University of Groningen have now solved one of these mysteries, however, and published their results in Nature Structural and Molecular Biology.

It is the mystery of the empty elevator. Many transporter molecules work like elevators: they move up and down through the cell membrane picking up their cargo and delivering it to the other side. After this ‘elevator’ has made its delivery, it needs to return to its original position to pick the next consignment.

‘The mystery is what is inside the elevator when it goes back’, explains biochemistry professor Dirk Slotboom. As Aristotle noticed, nature abhors a vacuum. ‘So what fills the space in the transporter once the cargo has been released?’ The way to find out is to investigate the proteins that make up a transporter, and determine its structure in the absence of cargo.

This is easier said than done. Slotboom: ‘To study the structure, you first have to grow a crystal from the protein, and subject it to X-ray diffraction. But as the cargo stabilizes the protein, it is difficult to grow crystals without it.’ This means that the structures of some transport proteins are known, but only with the cargo they transport.

It took the Slotboom group nine years to get the right crystals. ‘We study a protein that transports aspartate in microorganisms, but an analogous protein is active in the human brain, where it clears the neurotransmitter glutamate from the synapses.’

Their strategy was to try a great many homologous transport proteins from different microorganisms in the hope of finding one that would produce good crystals. They finally struck gold when PhD student Sonja Jensen managed to grow crystals from the aspartate transporter isolated from the prokaryote Thermococcus kodakarensis.

‘Once you have good crystals, you are very likely to have the structure. That’s when you break out the champagne. And then you have a thousand new questions’, says Slotboom. The structure revealed what fills the empty elevator: the side chain of an arginine amino acid positioned near the end of the protein that forms the transporter.

‘The side chain clicks into place inside the cell when the aspartate is removed. And there are probably a few water molecules in there as well.’ This explains an observation made in mutant versions of the brain transporter. ‘If this particular arginine is replaced by a smaller cysteine amino acid, the elevator can’t go up without the cargo, glutamate.’ This is because the cysteine lacks the side chain that fills the empty space. ‘So the lift will only go up with glutamate inside, meaning that the net transport is zero.’

This fundamental research on a transport protein has wide-ranging implications because it provides a better understanding of transport and what can go wrong. ‘One issue that comes to mind is the damage a stroke can cause’, explains Slotboom.

If part of the brain is starved of oxygen after a stroke, the transporters malfunction. Normally, the glutamate transporter removes glutamate from the extracellular space against a concentration gradient. But without oxygen, the energy to do this is no longer present. ‘The glutamate transport then effectively goes into reverse.’ This results in a high concentration of the neurotransmitter in the synaptic cleft, which causes a widespread activation of neurons. ‘And this actually causes cell death in the brain.’ If a small molecule could be designed to block the elevator, this might prevent some of the damage a stroke causes, if administered quickly.

But this is something for the long term, and not the direction Slotboom wants to follow with his research. ‘Our aim is to elucidate the mechanism of these transporters. We have discovered what fills the empty space in the elevator. Now, we want to find out exactly how it moves!’