3D modeling of the resting phase of the SusC-TonB-dependent transport system in Bacteroides thetaiotaomicron

Bacteroides are a dominant component of the human gastrointestinal tract, where gene clusters known as polysaccharide utilization loci (PULs) carry out the digestion and absorption of complex dietary polysaccharides. These systems rely on outer membrane SusC-like TonB-dependent transporters (TBDTs), which import substrate molecules utilizing the proton motive force generated by the inner membrane TonB protein complex. SusC-like TBDTs in Bacteroides possess a structurally distinct N-Terminal Extension (NTE) domain preceding the TonB-box, which is known to be crucial for interacting with the C-terminal domain of TonB. While previous X-ray crystallography studies of SusC-like TBDT have identified the key residues involved in glycan binding, the dynamic stability of these interactions within a fully assembled, full-length transporter is yet unknown. In this work, we first constructed a complete three-dimensional model of the resting-phase of the SusC-NTE interacting with the TonB-CTD, with the Fructo-oligosaccharide (FOS) substrate placed at the crystallographic binding site. The full-length SusC-NTE/TonB-CTD complex was then embedded within an asymmetric Bacteroides outer membrane and subjected to all-atom molecular dynamics (MD) simulations performed in triplicate (3 × 500 ns). RMSD and RMSF analyses, which superimpose closely across the independent replicates, validated the complex’s overall stability while also capturing the simultaneous flexibility of the extracellular loops and the conformational dynamics of the NTE domain. The FOS substrate was not retained at the crystallographically resolved binding site over the extended trajectories, in a construct lacking the SusD lid and with a substrate reported to have no measurable affinity for the SusCD complex, so that the production simulations describe the transporter in its unbound resting state. The plug domain occluded the β-barrel throughout all three replicates, at no point widening enough to admit a water molecule, so that movement through the SusC channel depends on the external force delivered by TonB under the proton motive force. Furthermore, Principal Component Analysis (PCA) highlighted reproducible exchange between two conformational states of the highly flexible N-terminal region, occurring in two of the three replicates at different times and without monotonic drift. Collectively, our study provides atomistic insight into the resting-state architecture of a full-length SusC transporter and enhances our understanding of the molecular processes underlying carbohydrate uptake in Bacteroides .