Nucleotide-sugar transporters (NSTs) are critical components of the cellular glycosylation machinery. They transport nucleotide-sugar conjugates into the Golgi lumen, where they are used for the glycosylation of proteins and lipids, and they then subsequently transport the nucleotide monophosphate byproduct back to the cytoplasm. Dysregulation of human NSTs causes several debilitating diseases, and NSTs are virulence factors for many pathogens. Here we present the first crystal structures of a mammalian NST, the mouse CMP-sialic acid transporter (mCST), in complex with its physiological substrates CMP and CMP-sialic acid. Detailed visualization of extensive protein-substrate interactions explains the mechanisms governing substrate selectivity. Further structural analysis of mCST’s unique lumen-facing partially-occluded conformation, coupled with the characterization of substrate-induced quenching of mCST’s intrinsic tryptophan fluorescence, reveals the concerted conformational transitions that occur during substrate transport. These results provide a framework for understanding the effects of disease-causing mutations and the mechanisms of this diverse family of transporters.
eLife digest The cells in our body are tiny machines which, amongst other things, produce proteins. One of the production steps involves a compartment in the cell called the Golgi, where proteins are tagged and packaged before being sent to their final destination. In particular, sugars can be added onto an immature protein to help to fold it, stabilize it, and to affect how it works. Before sugars can be attached to a protein, they need to be ‘activated’ outside of the Golgi by attaching to a small molecule known as a nucleotide. Then, these ‘nucleotide-sugars’ are ferried across the Golgi membrane and inside the compartment by nucleotide-sugar transporters, or NSTs. Humans have seven different kinds of NSTs, each responsible for helping specific types of nucleotide-sugars cross the Golgi membrane. Changes in NSTs are linked to several human diseases, including certain types of epilepsy; these proteins are also important for dangerous microbes to be able to infect cells. Yet, scientists know very little about how the transporters recognize their cargo, and how they transport it. To shed light on these questions, Ahuja and Whorton set to uncover for the first time the 3D structure of a mammalian NST using a method known as X-ray crystallography. This revealed how nearly every component of this transporter is arranged when the protein is bound to two different molecules: a specific nucleotide, or a type of nucleotide-sugar. The results help to understand how changes in certain components of the NST can lead to a problem in the way the protein works. Ultimately, this knowledge may be useful to prevent diseases linked to faulty NSTs, or to stop microbes from using the transporters to their own advantage.