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

Abstract

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

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.…”

“…6,7 The atomic structure of OxlT has been recently determined in the ligand-free outward-open and oxalate-bound occluded conformations (Figures 1A and 1B). 4 However, despite structural analysis and molecular dynamics (MD) simulation, the inward-open conformation of OxlT has been missing.…”

“…Our previous study identified the periplasmic and cytoplasmic gates which block the water influx and the exit of oxalate in the occluded state. 4,22 We here used the Cα-Cα distance between T38 and V240 in the periplasmic gate and the distance between M128 and P332 in the cytoplasmic gate as a measure to distinguish the conformational states of OxlT (Figure 1A). As shown in Figure 1C, the occluded conformation was stable in the conventional MD simulation, with no transition to the inward-open conformation observed.…”

“…The occluded conformation is stabilized by direct interdomain interactions as well as indirect interdomain interactions through the bound oxalate. 4 To facilitate the transition to the inward-open conformation, we weakened the former contribution in the cytoplasmic side, i.e., the direct interdomain interaction involving the cytoplasmic gate, by using the Gaussian accelerated 7 MD (GaMD) technique. 12 The GaMD is an accelerated MD method that assists the system in escaping from local minima by adding harmonic boost potential to user-selected energy terms (see Computational Methods section in the Supporting Information for details).…”

“…12,[23][24][25] We here applied the boost potential to the cytoplasmic-side interactions between residues 128-139 of the C-terminal domain and residues 332-348 of the N-terminal domain, in which some hydrophobic and electrostatic contacts involving the cytoplasmic gate were observed. 4 Orange points in Figure 1C show the distribution of the gate distances for 500-ns-long GaMD simulations (called "oxalatebound GaMD-1" here). The distance of the cytoplasmic gate was increased by applying the boost potential as expected, although the distance was intermediate between the occluded conformation and the previously reported inward-open conformation of the other MFS-type transporter NarK.…”