Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
- Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, New Zealand
- Department of Biochemistry and Biophysics, Stockholm University, Sweden
- Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Italy
- Biological Sciences and Biomedical Engineering, Bindley Bioscience Center, Purdue University West Lafayette, United States
- Department of Chemistry and Molecular Biology, Biochemistry and Structural Biology, University of Gothenburg, Sweden
- Centre for Antibiotic Resistance Research (CARe) at University of Gothenburg, Sweden
- Biomolecular Interaction Centre, Digital Life Institute, Maurice Wilkins Centre for Molecular Biodiscovery, and School of Biological Sciences, University of Auckland, New Zealand
- Biochemistry Department, School of Biomedical Sciences, University of Otago, New Zealand
- ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio Molecular Science and Biotechnology Institute, Department of Biochemistry and Pharmacology, University of Melbourne, Australia
- Department of Chemistry and Biochemistry, University of Montana, United States
- Department of Chemistry and Biochemistry, University of Lethbridge, Canada
- Biological Sciences Division, SLAC National Accelerator Laboratory, United States
- Department of Structural Biology, Stanford University School of Medicine, United States
- School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Australia
Figures
Figure 1
An overview of Neu5Ac metabolism in H. influenzae.
(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 …
Figure 2 with 3 supplements
The structure of HiSiaQM.
(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), …
Figure 2—figure supplement 1
Size-exclusion chromatography traces of SiaQM transporters suggests that HiSiaQM exists as multiple species in detergent.
(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) …
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Figure 2—figure supplement 1—source data 1
Original files for the gel and western blot analysis in Figure 2—figure supplement 1b and c.
- https://cdn.elifesciences.org/articles/92307/elife-92307-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
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.
- https://cdn.elifesciences.org/articles/92307/elife-92307-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Cryo-electron microscopy (cryo-EM) workflow for structure determination.
The two classes were determined as 36% of the particles (antiparallel dimer) and 36% (parallel dimer).
Figure 2—figure supplement 3
Representative density of the helices of HiSiaQM.
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 …
Figure 3 with 4 supplements
HiSiaQM self-association in lauryl maltose neopentyl glycol (L-MNG) and amphipol.
(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) …
Figure 3—figure supplement 1
HiSiaQM self-association in dodecyl-β-d-maltoside (DDM).
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 …
Figure 3—figure supplement 2
HiSiaQM self-association in lauryl maltose neopentyl glycol (L-MNG) (interference analysis).
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 …
Figure 3—figure supplement 3
HiSiaQM self-association in dodecyl-β-d-maltoside (DDM) (interference analysis).
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 …
Figure 3—figure supplement 4
Solubilisation of HiSiaQM in nanodiscs.
(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 …
Figure 4 with 2 supplements
Phospholipids bound to HiSiaQM.
(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 …
Figure 4—figure supplement 1
Protein sequence alignment of tripartite ATP-independent periplasmic (TRAP) transporter QM-subunits.
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 …
Figure 4—figure supplement 2
Protein sequence alignment of tripartite ATP-independent periplasmic (TRAP) transporter QM-subunits.
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 …
Figure 5 with 1 supplement
Transport assays demonstrate that lauryl maltose neopentyl glycol (L-MNG)-solubilised HiSiaQM is functional.
(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 …
Figure 5—figure supplement 1
The Na+ and substrate-binding sites of HiSiaQM.
(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…
Figure 6 with 3 supplements
Sedimentation velocity analytical ultracentrifugation (AUC) analysis of the interaction between HiSiaQM and HiSiaP in lauryl maltose neopentyl glycol (L-MNG) detergent.
(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…
Figure 6—figure supplement 1
HiSiaP is monomeric.
(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 …
Figure 6—figure supplement 2
Modelling the HiSiaPQM complex.
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 …
Figure 6—figure supplement 3
Residues involved in scaffold stabilisation and the interaction with SiaP.
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 …
Figure 7
Subunit substitution transport assays.
(a) Transport was measured with subunit substitution of HiSiaPQM with the fused SiaPQM from A. actinomycetemcomitans (Aa) and the non-fused SiaPQM from P. profundum (Pp). Transport activity was …
Videos
Video 1
Three-dimensional variability analysis of our HiSiaQM reconstructions shows only very subtle motion at the dimer interface and does not show any global elevator motions in the final reconstruction.
Tables
Key resources table
| 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 |
Additional files
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Supplementary file 1
Cryo-EM data collection, processing, refinement, and validation statistics.
- https://cdn.elifesciences.org/articles/92307/elife-92307-supp1-v1.docx
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Supplementary file 2
Protein sequences of HiSiaQM and HiSiaP expressed and purified in this work.
- https://cdn.elifesciences.org/articles/92307/elife-92307-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92307/elife-92307-mdarchecklist1-v1.docx
