Abstract
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.
Highlights
Nucleotide-sugar transporters (NSTs) are products of the solute carrier 35 (SLC35) gene family in humans and are a critical part of the glycosylation machinery in all eukaryotes (Hadley et al, 2014; Ishida and Kawakita, 2004; Song, 2013)
We identified mouse CMP-sialic acid transporter (mCST), which is 91% identical to human CST, as a suitable candidate for structural studies after screening a large panel of NST orthologs
One obstacle that we encountered when characterizing this construct was our discovery that commercial stocks of CMP-sialic acid (CMP-Sia) contain approximately 10% CMP (Figure 1—figure supplement 3A), which has a ~ 100 fold higher affinity towards mCST (Figure 1A)
Summary
Nucleotide-sugar transporters (NSTs) are products of the solute carrier 35 (SLC35) gene family in humans and are a critical part of the glycosylation machinery in all eukaryotes (Hadley et al, 2014; Ishida and Kawakita, 2004; Song, 2013) They are responsible for transporting nucleotide sugars from the cytoplasm, where they are synthesized, into the Golgi lumen where they are utilized by glycosyltransferases to glycosylate proteins and lipids (Figure 1—figure supplement 1A) (Capasso and Hirschberg, 1984; Milla and Hirschberg, 1989; Tiralongo et al, 2006; Waldman and Rudnick, 1990). Some NSTs are potently inhibited by antiviral nucleoside analogs, which may form the
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call