Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota

Abstract

Oxalobacter formigenes is an oxalate-degrading bacterium in the gut microbiota that absorbs food-derived oxalate to use this as a carbon and energy source and thereby helps reduce the risk of kidney stone formation of the host animals 1–4. The bacterial oxalate transporter OxlT uptakes oxalate from the gut to bacterial cells and excrete formate as a degradation product, with a strict discrimination from other carboxylates that serve as nutrients 5–7. Nevertheless, the underlying mechanism remains unclear. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two different conformations, occluded and outward-facing states. The oxalate binding site contains two basic residues that form salt bridges with a dicarboxylate substrate while preventing the conformational switch to the occluded state without an acidic substrate, a ‘disallowed’ state for an antiporter 8, 9. The occluded ligand-binding pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the binding pocket are completely blocked by extensive interdomain hydrophobic and ionic interactions. Nevertheless, a molecular dynamics simulation showed that a flip of a single side chain neighbouring the substrate is sufficient to trigger the gate opening. The OxlT structure indicates the underlying metabolic interactions enabling favourable symbiosis at a molecular level.

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota

“…The MD-related data have been deposited in the Zenodo repository [https://doi.org/10.5281/zenodo. 7597686] 94 . Coordinates with the PDB IDs 1PW4 58 , 1XF4 59 , 5B3N 60 , 4U4W 95 , 4U4T 96 and amino acid sequences of OFA family (IPR026355) were used in this study.…”

Expression of Plasmodium Major Facilitator Superfamily Protein in Transporters-Delta Candida Identifies a Drug Transporter

“…Homology modelling of Pf MFS_DT was done using Prime application in Schrodinger Maestro Suite 2022-4, using chain_A of bacterial oxalate transporter (OxlT 8HPK_A) [23] as a template. The generated model was further refined for its loop refinement and energy minimization using the OPLS4 force field in Schrodinger Prime [24] (i) .…”

“…The protein sequence (457 amino acids; accession ID: O96186) was retrieved from the UniProt database [22]. NCBI Protein BLAST against the PDB database identified chain_A of bacterial oxalate transporter OxlT 8HPK_A [23] as the best template for homology modeling, with a sequence similarity of 28.6 % and gaps of 1.5 %. The three-dimensional crystal structure of OxlT 8HPK_A was retrieved from the RCSB PDB database for use as the template.…”

“…Red: residues with high consensus values (>90%); Blue: residues with low consensus values (>50%, <90%); and, Stars: residues associated with Na + -and lysolipid-binding. [23] as a template. The generated model was further refined for its loop refinement and energy minimization using the OPLS4 force field in Schrodinger Prime [24] (i).…”

Accelerated Molecular Dynamics and AlphaFold Uncover a Missing Conformational State of Transporter Protein OxlT

“…The oxalate transporter (OxlT), an oxalate:formate antiporter in Oxalobacter formigenes, is a key protein that carries oxalate inside and its product formate outside the bacterium. [2][3][4] Transporter proteins such as OxlT work by an alternating-access mechanism. 5 They switch their conformations between the outward-open and inward-open states to alternatingly expose the binding site to opposite sides of the membrane.…”