How the ‘Pac-man’ transporter works

Officially, it’s called the ATP-binding cassette (ABC) transporter, but you can also call it Pac-man. It snaps up nutrients and other vital compounds from the surroundings of a cell and transports its cargo across the cell membrane. University of Groningen scientists have for the first time visualized how this Pac-man works. Their results appeared in Nature Structural & Molecular Biology on 8 December.

ABC transporters are found in all kingdoms of life and exist in countless forms. The system is modular, with domains on the outside that snap up specific substances (nutrients) like Pac-man, a transmembrane domain that transports the substance into the cell, and a third domain inside that powers the transport process.

‘We want to know exactly how it works’, says biochemistry professor Bert Poolman. And that means looking at single transporters. ‘So far, most research has looked at these transport proteins in bulk, which means you can easily have a quadrillion (10¹⁵) individual transporters in your sample.’ This in turn means you can only look at average behaviour. ‘Compare it to looking at the New York marathon from a helicopter. You are able to measure the average speed of the crowd, but you can’t observe how an individual is performing.’

To elucidate the different steps in the transport process (substrate capture, translocation over the membrane, release inside the cells), you have to look at a single molecule, ‘because in bulk, important steps are averaged out’, Poolman explains.

On the outside, the ABC transporter has the ‘Pac-man’ domains, which snap up specific substances. ‘There were two hypotheses to explain how this happens’, says Poolman. The first, called conformation selection, argues that Pac-man opens and closes its beak like it does in the computer game, and keeps it shut only after the right substrate is captured. The second, called induced fit, says that the beak remains open until the right substance happens to enter.

The technique the University of Groningen scientists used to investigate the workings of Pac-man is called single-molecule FRET . The two ‘jaws’ of the Pac-man beak are labelled with two different fluorescent markers. The first is excited by a laser and emits green light. This green light excites the second marker, which then emits red light. ‘The trick is that the amount of red light is directly proportional to the distance between the green and red light sources.’

In other words, when the beak is open there is a relatively big gap between both markers so the red light is weak, while a closed beak gives a stronger red signal as both markers are closer together. ‘Using this technique, we saw that the Pac-man beak is open until a substrate enters.’ So the induced fit hypothesis appears to be correct.

But there were more observations: ‘Occasionally, the beak would close without any substrate inside. And in that case, the transporter shuts down for a while. Overall, this means the transporters are down for a considerable portion of the time when the substrate composition or concentration is sub-optimal.’

Also, it was found that a tighter fit between the Pac-man and the substrate resulted in slower transport across the membrane. ‘A strong binding means that it is difficult to release the substrate. The binding needs to be carefully balanced because if it is too weak you don’t catch enough substrate.’

So what can we do with all this information? ‘Some of these ABC transporters are vitally important for pathogens. Understanding the dynamics of transport means we can potentially interfere with it.’ One option is to design small molecules that will block the transport system, and the new paper lays the foundations for this type of application. ‘Our fundamental research has resulted in a detailed model of part of the transport process, but more work needs to be done.’

Getting this far is already quite an achievement. No fewer than eight scientists from two research institutes were involved in the study, not only specialists in single molecule visualization from the Zernike Institute for Advanced Materials (ZIAM), but also biochemists from the Groningen Biomolecular Sciences and Biotechnology Institute (GBB).

‘There are three first authors of this paper, who all contributed equally to the project. It took us two years to refine the single molecule measurements enough to get these results’, Poolman and his biophysics colleague Thorben Cordes explain. And, they add, ‘We have produced some of the best single molecule measurements ever made for membrane proteins and received excellent feedback from peers in the field.’

Reference: Giorgos Gouridis, Gea K Schuurman-Wolters, Evelyn Ploetz, Florence Husada, Ruslan Vietrov, Marijn de Boer, Thorben Cordes & Bert Poolman: Conformational dynamics in substrate-binding domains influence transport in the ABC importer GlnPQ (DOI: 10.1038/nsmb.2929)

Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.

Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands.