Structural insights into the inhibition of glycine reuptake

Structural insights into the inhibition of glycine reuptake

Glycine is the smallest amino acid - one of the building blocks of proteins. It acts also as a neurotransmitter in the brain, enabling neurons to communicate with each other and modulating neuronal activity. Many researchers have focused on increasing glycine levels in synapses to find an effective treatment for schizophrenia. This could be done using inhibitors targeting Glycine Transporter 1 (GlyT1), a protein that sits in neuronal cell membranes and is responsible for the uptake of glycine into neurons. However, the development of such drugs has been hampered because the 3D structure of GlyT1 was not known.

GlyT1 turned out to be particularly challenging to study, because it is unstable when extracted from the cell membrane. To stabilise it, scientists combined several approaches, such as creating more stable variants of the protein. To catch the transporter in a clinically relevant state, the team used a chemical created by Roche that binds and stabilises GlyT1 from the inside, and designed a synthetic mini-antibody (sybody) that binds it from the outside.

The scientists tested 960 different conditions and managed to obtain GlyT1 crystals in one of them. "The crystals were very small and difficult to image. We chose to measure them at EMBL Hamburg's beamline P14, which is well suited for challenging experiments like this one," says the author. The X-ray beam at P14 is particularly strong and focused, and its equipment has features tailored for work with even micrometre-sized crystals. Yet the quality of the crystals was variable, which made data collection challenging. Eventually, the authors perseverance paid off. "I remember when I saw electron density of the inhibitor for the first time. I was so excited, I couldn't sleep for two nights," the author says. "You live for those rewarding moments."

The final challenge was the data analysis. While the crystals were giving only weak diffraction patterns due to their small size, the strong X-rays destroyed the crystals in less than a second. A single crystal would yield only partial information about the structure, so the author had to collect data from hundreds of crystals.  Combining partial datasets was complex for the existing software, but the authors wrote software specifically designed for such cases. It enabled them to merge datasets into a full picture of GlyT1 at 3.4 Å resolution (1 Å, or ångström, is one ten-billionth of a metre - about the size of a typical atom).

The analysis revealed an unexpected structure of GlyT1. In contrast to other neurotransmitter transporters, which are bound by their inhibitors from the outer side of the cell membrane, GlyT1 is bound by its inhibitor from the inner side. "The structure was a surprise for us. It seems that the GlyT1 inhibitor must first cross the cell membranes before it can access GlyT1 from the inside of the neurons," says a senior author in the study.

"This structure provides a blueprint for developing new inhibitors of GlyT1, be they organic molecules or antibodies," explains the author. "The sybody developed for this study binds GlyT1 at a previously unknown binding site and locks it in a state in which it cannot transport glycine any more. We could use this knowledge to develop drugs targeting not only GlyT1, but also other membrane transport proteins in the future."