(1) H. influenzae is sialidase negative and relies on environmental sialidases to hydrolyse and release terminal Neu5Ac from human glycoconjugates. (2) Outer membrane porins facilitate diffusion of …
(a) Coulomb maps for the parallel (3.36 Å) and antiparallel (2.99 Å) HiSiaQM homodimers. The periplasmic surfaces of the monomers are facing the same direction for the parallel dimer (PDB: 8THI), …
(a) HiSiaQM purified in dodecyl-β-d-maltoside (DDM) (green trace) results in significant aggregation at 45–50 mL, which is not apparent when solubilised in lauryl maltose neopentyl glycol (L-MNG) …
Original files for the gel and western blot analysis in Figure 2—figure supplement 1b and c.
Image containing Figure 2—figure supplement 1b and c and original files for the gel and western blot analysis with highlighted bands and sample labels.
The two classes were determined as 36% of the particles (antiparallel dimer) and 36% (parallel dimer).
Clear side-chain densities are present for almost all of the residues of HiSiaQM. Density for the antiparallel dimer is shown as it is higher resolution and was used for the structural analysis in …
(a) Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of HiSiaQM in L-MNG (left panel). Two well-resolved species exist at 7.3S (diffusion coefficient, D = 4.8 × 10–7 cm2/s) …
Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of HiSiaQM in DDM (left panel). Two well-resolved species exist at 7.6S (diffusion coefficient, D = 5.0 × 10–7 cm2/s) and …
Sedimentation of HiSiaQM in L-MNG (left panel) with absorbance data (pink) and interference data (green). Two well-resolved species exist at 6.7S and 9.4S, with the smaller species constituting …
Sedimentation of HiSiaQM in DDM (left panel) with absorbance data (pink) and interference data (green). Two main species exist at ~4.5S and ~6.2S, with the smaller species constituting greater than …
(a) The size-exclusion chromatogram following nanodisc reconstitution with a 1:4:80 ratio of HiSiaQM:MSP:lipid identifies the presence of multiple species. Three fractions across the elution profile …
(a) The dimer has well-defined areas of density (grey) that correspond to bound phospholipids. Two mechanistically important areas are the dimer interface and fusion helix pocket. (b) Phospholipids …
Protein sequences were obtained from the National Center for Biotechnology Information, aligned using Kalign (Lassmann, 2019) and coloured in Jalview (Waterhouse et al., 2009) with Clustal X …
Protein sequences were obtained from the National Center for Biotechnology Information, aligned using Kalign (Lassmann, 2019) and coloured in Jalview (Waterhouse et al., 2009) with Clustal X …
(a) [3H]-Neu5Ac uptake was measured at multiple time intervals under each condition and used to calculate transport rates. HiSiaQM had the highest activity in the presence of HiSiaP, a membrane …
(a) Closeup views of the cryo-electron microscopy (cryo-EM) density (blue) at the Na1 and Na2 sites of the antiparallel HiSiaQM dimer. Density is present for both Na+ ions (green spheres). Na+-bindin…
(a) Titrating increasing concentrations of HiSiaQM (blue, 40 µM [2.88 mg/mL]; pink, 20 µM; green, 10 µM; brown, 5 µM; other concentrations omitted for clarity) against fluorescently labelled FITC-HiS…
(a) Analytical ultracentrifugation experiments of HiSiaP (expressed in the periplasm) without Neu5Ac or with 5 mM Neu5Ac resulted in sedimentation coefficients of ~2.8S without Neu5Ac (blue) and …
AlphaFold2 was used to model the complex of two HiSiaP monomers (PDB: 3B50) bound to the parallel HiSiaQM dimer from this work (PDB: 8THI). The HiSiaQM monomers are shown in two shades of green and …
The resolution of the HiSiaQM structure (coloured as in Figure 2c) shows the positions of R30, S356, E429, and R484 as viewed from the periplasm. R30 (SiaQ, helix 1) forms a salt bridge with E429 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Haemophilus influenzae Rd KW20) | SiaP; SiaQM | UniProt | P44542, P44543 | |
Strain, strain background (Escherichia coli) | BL21 (DE3); TOP10 | Invitrogen | Chemically competent cells | |
Antibody | Anti-Xpress IgG (mouse monoclonal) | Invitrogen | WB (1:2500) | |
Antibody | Anti-mouse IgG (rabbit) | Sigma-Aldrich | WB (1:15000) | |
Recombinant DNA reagent | pET22b(+) (plasmid) | GenScript | Provides pelB leader sequence | |
Recombinant DNA reagent | pBAD/HisA (plasmid) | Thermo Fisher Scientific | ||
Peptide, recombinant protein | cNW11 | Now Scientific | Covalent nanodisc protein |
Cryo-EM data collection, processing, refinement, and validation statistics.
Protein sequences of HiSiaQM and HiSiaP expressed and purified in this work.