Abstract
Human glucose transporter 9 (hSLC2A9) is critical in human urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 & SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for urate binding. Our functional studies confirmed that N429 is a key residue for both urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating urate transport. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.
Highlights
Our earlier studies have revealed that a single hydrophobic residue in transmembrane helix 7 (H7), is a major determinant of substrate selectivity in the hGLUTs10,11
Most of these residues are conserved in SLC2A9 and SLC2A5, sequence alignment reveals residues that may play important roles in the distinct urate transport abilities of SLC2A9
We questioned whether fructose/urate binding sites are conserved or different between SLC2A9 and SLC2A5. If their binding sites are very similar, do the non-conserved residues within the putative binding pocket play a specific role in urate binding, or does urate bind to distinct sites that differ from the ones for fructose ? To address these questions and identify key residues, we performed in silico docking studies to examine both fructose and urate binding sites utilizing the molecular modeling of hSLC2A9b based on the SLC2A5 crystal structure. hSLC2A9b has an identical amino acid sequence to the full-length hSLC2A9, yet has a shorter N-terminus that is predicted to reside outside of the membrane and not influence transport, but instead targets the two isoforms to different membrane domains within epithelial cells[17]
Summary
Wentong Long[1], Rashmi Panigrahi[2], Pankaj Panwar[2], Kenneth Wong[1], Debbie O′Neill[1], Xing-Zhen Chen[1], M. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5) These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket. Most of these residues are conserved in the fructose binding pocket in our hSLC2A9 model based on this SLC2A5 crystal, except Gln[287] (Tyr[298] in hSLG2A9) and His[418] (Asn429) (Fig. 1) We reasoned that these two residues might be critical for hSLC2A9 to handle urate and fructose transport. Our SLC2A9 molecular docking studies based on the newly reported SLC2A5 crystal structure suggest that urate binds within the same translocation pocket as fructose; the two substrates interact with some distinct residues. This study provides strong evidence that urate and fructose transport mediated by hSLC2A9 involves multiple structural elements that together provide a unique ability to handle these two very different substrates
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