Researchers have succeeded in determining the 3D structure of the enzyme that attaches sugar chains to proteins. Whenever cells receive signals, interact with other cells or identify viruses and bacteria, the process involves not only proteins but also sugar chains attached to their surface.

The subject of relatively little attention until now, these structures differ widely in composition and branching and seem to help ensure that proteins fold correctly and are able to perform their specific tasks.

Several observations demonstrate the importance of the sugary appendages: identical proteins with different sugar chains have different functions, diseased and healthy cells have differing sugar compositions on their surfaces, and drugs to which they are attached are tolerated better.

The emerging research field of glycobiology deals with biosynthesis, structure and the diverse functions of the sugary appendages. They have determined the three-dimensional structure of oligosaccharyltransferase (OST) in yeast. This is the enzyme that connects proteins to sugar trees.

Made possible thanks to cryo-electron microscopy

Determining the structure of OST was not easy: to start with, Jilliane Eyring, the third lead author of the report, modified the yeast cells so that the enzyme could be targeted and purified. Wild first had to extract the enzyme, which is embedded in a membrane in the cell, from large quantities of these yeast cells and then purify it in a laborious procedure. 

Cryo-Electron Microscope

 The OST molecules were applied to a small grid, flash-frozen as an individual, separate particles and imaged using a high-resolution cryo-electron microscope.

This gave rise to thousands of images showing the enzyme complex from every possible viewing angle. From these images, Julia Kowal, the second lead author, generated the three-dimensional structure of OST. It took six weeks to perform the calculations on the computer cluster. The result was an electron density map which shows the enzyme as an"electron cloud."

An opportune moment

They were able to benefit from the "resolution revolution" currently taking place in cryo-electron microscopy and make use of a state-of-the-art device with atomic resolution.  To interpret the electron microscopy data, Wild had to fit the amino acid sequence of OST "manually" into the small clouds in the three-dimensional map.

Thus, the researcher was able to depict the 3D structure of OST in full detail a breakthrough that was met with delight among researchers after it was published online. They had expected the catalytic unit to be in the middle of the enzyme. Surprisingly, however, it's on the outside, and the shape is reminiscent of a wide-open mouth.

A good model for human enzymes

The researchers draw this conclusion from the fact that yeast OST's active center, where the proteins are fitted with sugars, closely resembles the corresponding area in bacteria. 

Active Center 

This means that the active center has barely changed over the course of evolution, which means there's a good chance that it still works in a very similar way in mammals, and therefore also in humans.