Proposed mechanism for Rft1-mediated scrambling of a dolichol-linked oligosaccharide
Description
Co-folding models of the Rft1 and MurJ transporter in complex with the substrates, M5-DLO and lipid II respectively. Data used in preprint https://doi.org/10.64898/2025.12.07.692794
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Steps to reproduce
The structure prediction algorithms Alphafold 3 (installed locally) and Chai1 (accessed through the web portal) were used to predict alternate conformations of Rft1 and the Rft1/M5-DLO complex. The initial structure of M5-DLO was download form the Chemical Entities of Biological Interest data base, entry CHEBI:132515 and converted to SMILE format. Instead of the typical 14-19 isoprenyl repeats for yeast M5-DLO, a shortened version with four of these was used to simplify the docking analysis. Our choice of a short C20 isoprenoid to model M5-DLO is supported by recent data showing that Rft1 can scramble Man5GlcNAc2-PP-phytanol which contains a C20 chain. Chai1 was run without multiple sequence alignment and no constraints, producing the default five Rft1/M5-DLO complex models. Residues <5 Å of the docked M5-DLO in each of the five Rft1/M5-DLO predictions output by Chai1 were determined using the Residue Interaction Network analyser and structureViz plugins in Cytoscape (v 3.10.3) and Chimera (v1.19). 5 seeds were used for the local AlphaFold3 run using M5-DLO in SMILES format as well generating at total of 25 Rft1/M5-DLO complex models. To enforce stereochemical accuracy, particularly at the critical lipid-glycan interface, a second AlpahFold3 run was perform where ligand topology was defined using the Chemical Component Dictionary (CCD) format linked via the bondedAtomPairs syntax, rather than SMILES strings, which have been shown to frequently misassign anomeric configurations in complex glycans. The M5-DLO ligand was assembled using octaprenyl pyrophosphate (CCD code: OTP) as a structural analog for the dolichol carrier. The glycan moiety was constructed from standardized monosaccharide building blocks. To recapitulate the native biosynthetic linkage, N-acetyl-a-D-glucosamine (CCD code: NDG) was explicitly specified as the reducing-end residue connected to the pyrophosphate, preserving the biological a-anomeric configuration distinct from protein N-glycosylation. This was extended by N-acetyl-b -D-glucosamine and b-D-mannose to form the core, with subsequent b-D-mannose branches defined via explicit atom-pair bonding to match the canonical M5 topology. Modeling was performed using three independent random seeds. The correct stereochemistry of the docked M5-DLO in all the 15 complex models were assessed and confirmed to be realistic using the Privateer online tool.
Institutions
- Universite Libre de Bruxelles