Structure and Biochemical Characterization of the Mammalian Fructose Transporter GLUT5

Abstract

Excessive consumption of fructose in the Western diet has been associated with metabolic disorders such as type 2 diabetes and obesity. Altered expression and activity of the fructose uniporter GLUT5, a member of the family of GLUT transporters that facilitate the diffusion of monosaccharides across membranes, has been linked to such disorders. Using Saccharomyces cerivisiae as an expression host, and fluorescence-based methods, we identified mammalian GLUT5 orthologues that are suitable for biochemical and structural studies. Here, we present a crystal structure of GLUT5 from Bos Taurus (bovine) in an inward-facing conformation, refined against data extending to ∼ 3.1 A resolution. Like GLUT1, GLUT5 shows the typical Major Facilitator Superfamily (MFS) fold, which consists of two 6-TM bundles, and four additional helices that form a soluble domain on the cytoplasmic side of the membrane. The substrate-binding site is highly similar to that observed in the recent crystal structure of human GLUT1, and to those of other GLUT isoforms based on amino acid sequence. However, there are notable differences. In particular, the substrate-binding site of GLUT5 is larger, because the equivalent of a tryptophan residue lining the cavity that contains the substrate-binding site in GLUT1 is an alanine in GLUT5. Furthermore, we have identified a single point mutation that switches the substrate preference of GLUT5 from D-fructose to D-glucose. Overall, our structural and biochemical data provide novel insights into the structure and substrate specificity of GLUT5, a member of the family of the medically relevant GLUT transporters.