Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9)

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.

Large-scale whole-exome sequencing association studies identify rare functional variants influencing serum urate levels

“…We found both the extracellular and intracellular interfaces to be enriched with variants of large effect size, strongly supporting the role of glycosylation at N61 as critical for GLUT9 function. In addition we found that the four core variants, F88L, V184M, V428I, and N433S each disrupted predicted hydrogen bond interactions between urate and four key urate-binding residues (Y52, Q299, G402, and N429 28 , 41 ), changes that may alter the substrate binding kinetics and affinity.…”

“…A group of particularly high interest includes the variants mapping to the predicted core helices (F88L; V184M; V428I; N433S). Previously, Long et al 28 . defined four residues, Y42, Q299, G402, and N429, as forming hydrogen bond interactions with urate in the binding pocket (with the N-O bond distance as being the typical 3.0 Å; Fig.…”

“…SLC2A transporter NCBI Reference Sequences were obtained from the NCBI and aligned using MacVector (MacVector INC, USA). The structural model of human SLC2A9b was constructed in a manner similar to the previously published Long et al 28 . The Wt and variant protein sequence were submitted separately to the I-Tasser Server at the University of Michigan using the recent crystal structure of rat SLC2A5 (PDB:4YBQ) as the template 66 , 67 .…”

“…The resulting model had a confidence score (C-score) of +0.42 (scale −5 to +2), indicating a model with a relatively high confidence based on template alignment and structural similarity. The modeling of urate as a substrate and the SLC2A9b binding pocket with predicted hydrogen bonding interactions was based on published docking studies by Long et al, and putative damaging variants with urate-associated betas < −1 and > 1 were mapped onto the hSLC2A9 model (Supplementary Data 24 ) 28 .…”

Target genes, variants, tissues and transcriptional pathways influencing human serum urate levels

“…Of note, the 3.5% of individuals in the three highest GRS categories had a >3-fold increase in the risk of gout compared to individuals in the most common GRS category. This risk is comparable to a monogenic disease of modest effect size 31 , but affects a comparatively higher proportion of the population.…”