Fig. 3. Analysis of the putative peptidoglycan binding site within the SLH domain.

a Binding assay of recombinant full-length and barrel-only VpOmpM1 to sacculi (PG) isolated from V. parvula and E. coli. After incubation of protein with PG, the insoluble PG was pelleted by centrifugation, washed by resuspending in buffer, and pelleted again. The supernatant from the binding reaction (S), the wash (W) and the resuspended PG pellet (P) were boiled and analysed by SDS-PAGE. Three independent binding experiments were performed and yielded similar results. b Close-up view of the VparOmpM1 SLH domains predicted by AlphaFold2, the crystal structure of SLH domains 1–3 from Bacillus anthracis Sap (surface array protein) (PDB 3PYW)⁴⁴, and the crystal structure of SLH domains 1–3 from Paenibacillus alvei SpaA in complex with the monosaccharide 4,6-pyr-β-D-ManNAcOMe (space-filling representation) (PDB 6CWH)⁴⁵. Views were generated from a superposition. c Multiple sequence alignment results from different subsets of SLH domain-containing proteins mapped onto the sequence of VpOmM1 SLH domain and coloured by conservation using ConSurf¹⁰⁴ (colour key). Row 1—OmpM homologues from Negativicutes; row 2—representative OmpM homologues from all diderm Firmicutes; row 3—representative OmpM homologues from diderm Terrabacteria, excluding Firmicutes; row 4—SLH domains from non-OmpM from diderm Firmicutes. The arrows point to Y36 and Y65, which could be important for binding polyaminated PG. d ConSurf results mapped onto the predicted SLH domains of VpOmpM1 (same sequence subsets and colour key as b). Arrows point to the grooves between SLH protomers. Y36 is shown in space-filling representation. The datasets used for ConSurf analysis correspond to the datasets used in a previous study¹⁶ that were subsampled using custom scripts. e Conserved residues shown as stick models on the predicted SLH structure, coloured by chain, except for Y36 and Y65 which are in purple. SpaA bound to monosaccharide (PDB 6CWH) was superposed onto the VpOmpM1 SLH structure; the monosaccharide is in grey, the SpaA protein model is not shown.