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Nature CommunicationsBiochemistryPeer-reviewed journal article

Mechanisms of urate transport and uricosuric drugs inhibition in human URAT1

Wenjun GuoiD, Miao WeiiD, Yunfeng LiiD, Jiaxuan XuiD, Jiahe ZangiD, Yuezhou CheniD, Lei CheniD

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Abstract

High urate levels in circulation lead to the accumulation of urate crystals in joints and ultimately inflammation and gout1. The reabsorption process of urate in the kidney by the urate transporter URAT1 plays a pivotal role in controlling serum urate levels2. Pharmacological inhibition of URAT1 by uricosuric drugs is a valid strategy for gout management3. Despite the clinical significance of URAT1, its structure and mechanism remain elusive. Here, we report the structures of human URAT1 (hURAT1) in complex with substrate urate or inhibitors benzbromarone and verinurad at resolution ranges from 3.0 to 3.3 [A]. Urate-bound hURAT1 adopts the outward-facing conformation. Urate is wrapped in the center of hURAT1 by five phenylalanines and coordinated by two positively charged residues on each side. Uricosuric compounds benzbromarone and verinurad occupy the urate-binding site of hURAT1 in the inward-facing conformation. Structural comparison between different conformations of hURAT1 reveals the rocker-switch-like mechanism for urate transport. Benzbromarone and verinurad exert their inhibitory effect by blocking not only the binding of urate but also the structural isomerization of hURAT1.

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (9 Sept 2024) · CC BY 4.0

Cryo-EM structures of human URAT1 with urate, benzbromarone, or verinurad (3.0 to 3.3 Å) reveal a rocker-switch transport cycle and inhibitors that lock the inward-facing state.

Urate reabsorption by URAT1 controls serum urate and is a validated gout drug target, yet its structure and transport mechanism were unresolved. These outward- and inward-facing structures link substrate binding and drug inhibition to conformational switching.

Key results

  1. ShownUrate-bound hURAT1 is outward-facing. Urate sits in a cage of five phenylalanines (F241, F360, F364, F365, F449) and is contacted by R477 and K393; alanine mutants of the cage residues impair uptake.

    Urate map at 3.34 Å. F364A and F449A nearly abolish urate uptake; F360A differs from hURAT1WT at P = 0.0003 (n = 3 technical replicates). · The urate binding site on hURAT1; Cryo-EM image processing; Figure legends

  2. ShownBenzbromarone and verinurad both occupy the urate site in an inward-facing state with highly similar binding-site architecture. The F360T substitution reduces the potency of both inhibitors while keeping uptake activity similar to wild type.

    Maps at 2.99 Å (benzbromarone) and 3.21 Å (verinurad); RMSD 0.95 Å between the two structures. F360T uptake not significantly different from WT (P = 0.2156 in the benzbromarone panel, 0.149 in the verinurad panel) but potency reduced. · The binding mode of benzbromarone; The binding mode of verinurad; Cryo-EM image processing; Figure legends

  3. ShownComparing the outward- and inward-facing structures shows a rigid-body, rocker-switch motion of the C-terminal domain relative to the N-terminal domain. Disrupting interdomain polar pairs lowered urate uptake for R465A, R284A and the K145A+R487A double mutant, but not R487A alone.

    R465A and R284A sharply reduce uptake (R284A P = 0.0003). The K145A+R487A double mutant reduces uptake (P = 0.001) whereas R487A alone does not (P = 0.9935); n = 3 technical replicates. · Conformational changes of hURAT1 between states; Figure legends

  4. SuggestedSuperposing the inhibitors onto the outward-facing structure produces steric clashes, so they likely block urate binding and also trap the transporter in the inward-facing conformation.

    Benzbromarone clashes with F449 and verinurad clashes with R447 in the outward-facing model; this is a structural superposition, with no direct kinetic test of state locking. · Inhibition mechanism of hURAT1; Discussion

  5. ShownA consensus-designed construct (hURAT1EM) with peripheral mutations preserved urate transport and inhibitor sensitivity while improving expression and stability, which enabled the structures.

    15 mutations among 553 residues (97% identity to WT); higher surface expression (P = 0.0012, two-tailed t test, n = 3 technical replicates); thermostability improved (P < 0.0001). · Structural determination of hURAT1; Consensus design; Figure legends

Methods, data and reproducibility

A consensus-designed hURAT1 construct in lipid nanodiscs was imaged by single-particle cryo-EM with urate, benzbromarone, or verinurad. Structure-guided mutants were tested for urate uptake, surface expression, and inhibitor IC50 in cells.

  • Cryo-EM map and model of urate-bound outward-facing hURAT1EM in nanodiscs · human (hURAT1 transporter) · EMD-60823; PDB 9IRW
  • Cryo-EM map and model of benzbromarone-bound inward-facing hURAT1EM · human (hURAT1 transporter) · EMD-60824; PDB 9IRX
  • Cryo-EM map and model of verinurad-bound inward-facing hURAT1EM · human (hURAT1 transporter) · EMD-60825; PDB 9IRY
  • Urate uptake, surface expression and inhibitor IC50 assays of hURAT1 variants in FreeStyle 293F cells · human (hURAT1 transporter) · n = n = 3 technical replicates; repeated independently twice or three times
  • SLC22A12 family sequences from UniProt used for consensus design · n = 120 sequences
  • Mice immunized with hURAT1-expressing cells for selection of the Fab15 antibody · mouse (BALB/c)
  • Template for homology model building (rOAT1) · not stated in text (named rOAT1) · PDB 8SDU
Methods & tools
cryoSPARC, MotionCor2, Topaz, Coot, PHENIX, SWISS-MODEL, UCSF Chimera, ChimeraX, MUSCLE, AlphaFold2 database, GraphPad Prism v.6, IgBlast
Reagents & environment
GraphPad Prism v.6 for IC50 fits; other software versions and containers not stated.
Reproducibility gaps
No code repository is given. Maps and models are deposited in EMDB and the PDB, and source data are said to accompany the paper.
Limitations (4)
  • The urate-bound structure may represent an outward-facing partially occluded state, because M36 partly blocks the extracellular entrance pathway. · stated by the authors
  • Several mutants (M36T, F241S, Q473A, F364L, F449L) had significantly reduced urate uptake activity, so their effects on benzbromarone or verinurad inhibition could not be analyzed confidently. · stated by the authors
  • The rocker-switch and inhibitor-locking model rests on static structures, each captured with a different ligand, and on mutants tested by uptake assays with n = 3 technical replicates. No dynamics or kinetics directly test the state transitions. · noted in this summary
  • All structures use the engineered hURAT1EM construct rather than wild-type hURAT1. Functional similarity was shown by activity and inhibition assays, but no wild-type structure was determined to confirm identical conformations. · noted in this summary

Read this if you study SLC22 or MFS transporter mechanisms, or want structural details of how uricosuric drugs bind URAT1 for gout drug design.

Citations
35
FWCI
21.83
Field percentile
top 1%

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