Overview of common PGT family members. Sm-monoPGTs do occupy one leaflet of the membrane, comprise only the catalytic core of monoPGTs (blue surface) and are exemplified by the structurally characterized PglC from Campylobacter concisus (dark blue cartoon). Lg-PGTs feature the C-terminal conserved catalytic core domain (blue surface) and two uncharacterized N-terminal domains. These domains feature a predicted transmembrane helix bundle (purple surface) and a domain of unknown function (gold surface). Poly-PGTs such as MraY from Aquifex aeolicus (magenta cartoon) comprise a catalytic domain, which is structurally distinct Sm- or Lg- PGTs and have been demonstrated to dimerize in their active form.

Summary of the differences between PGT families

Characterization of S. enterica WbaP in SMALP. A. WbaP in SMALP analyzed by SEC-MALS. A small peak is observed at the void volume of the column, while the main peak has a calculated molecular weight of 255 ± 8 kDa, with a polydispersity value of 1.001. B: WbaP in SMALP analyzed by mass photometry. The sample is monodisperse, and the main species has an apparent molecular weight of 239 ± 28 kDa. C. Mass spectrum of WbaP in SMALP analyzed by Direct Mass Technology mode. D. Western blot analysis of Lysine-reactive dithiobis(succinimidyl propionate) (DSP) crosslinker reacted with WbaP in SMALP. Lanes: 1: Control, 2: 0.1 mM DSP, 3: 0.25 mM DSP, 4: 1 mM DSP, 5: 5 mM DSP, 7: 0.1 mM DSP reduced, 8: 0.25 mM DSP reduced, 9: 1 mM DSP reduced, 10: 5 mM DSP reduced

AlphaFold predictions of S. enterica WbaP monomer and dimer. A: WbaP monomer prediction. B: WbaP dimer prediction. C: Close-up view of predicted dimer interface showing the interdigitating β-hairpin motifs between DUFA and DUFB. PGT domains hidden for clarity.

Probing the predicted interface of the WbaP dimer. A. and B. AlphaFold prediction with Cys pair variants modeled as sticks. The β-hairpin of one monomer is shown in sky blue and the continuing β- strand of the other monomer is shown in orange. C. Structure and crosslinking radii of thiol reactive crosslinkers. D. Crosslinking of Cys pair WbaP variants in SMALP using Dibromobimane (bBBr) and Cu Phenanthroline. E. bBBr crosslinking of single and double WbaP Cys variants in SMALP.

XLMS of WbaP in SMALP. A. Structures of amino reactive crosslinkers used for XLMS analysis. B. Overview of identified WbaP crosslinks from different protease treatments and crosslinkers. C. Identified crosslinks mapped onto S. enterica WbaP AlphaFold dimer model. DSS crosslinks shown in black, tBu-PhoX crosslinks shown in gray. D. Residues linked by intermolecular crosslinks. The distance between these residues within a single monomer and between monomers is shown.

Structure of S. enterica WbaP in SMALP at 4.1 Å. WbaP dimer colored in blue and orange. A. Unsharpened map displaying clear density for stabilizing liponanoparticle. Unsharpened density colored light gray. B. Sharpened map after masked local refinement. Sharpened density colored light gray C. Superposition of refined WbaP model and full-length AlphaFold prediction, RMSD: 2.83 Å. AlphaFold dimer colored gray. A predicted helix-turn-helix motif lacks density in the experimental map.

Comparison of S. enterica WbaP PGT domain and PglC from Campylobacter concisus (PDBid 8G1N). WbaP shown in light blue, PglC in magenta. A. Superposition of WbaP and PglC. While the overall RMSD is 1.83 Å, regions in PglC which can be used to computationally assign substrate are significantly different in WbaP. A loop region in PglC is replaced with a helix-turn-helix motif in WbaP, and the C-terminus of WbaP is significantly shorter than that of PglC. B. Top: an aromatic box motif in PglC is conserved among PGTs that utilize UDP-diNAcBac. Aromatic box residues are shown as dark green sticks. Bottom: Residues found in S. enterica WbaP at homologous positions to aromatic box residues in PglC shown as dark green sticks. Aromatic box residues are non-conserved in WbaP.

WbaP UMP titration assay. Left: Percent activity vs UMP concentration. A UMP-dependent decrease in WbaP activity is observed. Right: Curve-fitting of UMP inhibition data yields an IC50 apparent of 4.1 μM.