Mechanism of substrate binding and transport in BASS transporters
- School of Life Sciences, University of Warwick, United Kingdom
- Department of Physics, Arizona State University, United States
- Malvern Panalytical Ltd, United Kingdom
Figures
Figure 1 with 2 supplements
Structure of ASBTNM.
(A) Structure of ASBTNM in complex with taurocholate ASBTNM(TCH) (Hu et al., 2011). The panel domain is coloured salmon. The core domain is coloured blue with the cross-over helices, TM4 and TM9 in …
Figure 1—figure supplement 1
Sequence alignment.
Figure 1—video 1
Morph between ASBTNM(Pan) and ASBTNM(TCH).
The colouring is shown as in Figure 1. The sodium ions and pantoate from the ASBTNM(Pan) structure are held rigid during the morph of the protein atoms.
Figure 2
Pantoate binding to ASBTNM.
(A) Results from thermostability assay showing that pantoate stabilises ASBTNM to a similar extent to taurocholate. The compounds are shown below. The mean and standard deviations are shown based on …
Figure 3 with 1 supplement
Pantoate binding site.
(A) The pantoate binding site in the ASBTNM(Pan) structure, coloured as in Figure 1. Hydrogen bonds are shown as dashed lines. (B) 2mFo-Fc density for the refined structure. The density is contoured …
Figure 3—figure supplement 1
Sodium site for the ASBTNM(Pan) structure.
(A) Electron density associated with the sodium ions and pantoate. The 2mFo-DFc density (blue) was calculated based on phases from the refined structure and was contoured at 1σ. The mFo-DFc density …
Figure 4 with 1 supplement
Structure of ASBTNM without pantoate or taurocholate.
(A) Superposition of ASBTNM(ns) (wheat) on ASBTNM(TCH) (pale green) highlighting the similarity of the two structures. The main differences are in the position of TM1, where TM1a adopts a slightly …
Figure 4—figure supplement 1
Electron density associated with ASBTNM(ns).
(A) Electron density in the vicinity of the sodium ions. The 2mFo-DFc density was calculated based on phases from the refined structure and was contoured at 1σ. (B) There is additional electron …
Figure 5 with 1 supplement
Characterisation of pantoate binding to mutants of ASBTNM.
Pantoate binding to ASBTNM mutants measured by isothermal calorimetry.
Figure 5—figure supplement 1
Testing a panel of compounds for potential binding to ASBTNM.
(A) Compounds were subjected to the stability assay. These compounds include citrate, which has been observed in two different crystal structures of ASBTYF (Wang et al., 2021; Zhou et al., 2014), …
Figure 6 with 2 supplements
Molecular dynamics simulations.
(A) Hydrogen bonds between the pantoate and protein followed over the course of the simulations starting without (left) or with (right) sodium bound. Red, yellow, green, and blue indicate a contact …
Figure 6—figure supplement 1
Root mean square deviation (RMSD) of protein and pantoate during simulations.
(A) Pantoate heavy-atom RMSD relative to the initial structure for each repeat simulation starting without (i) or with (ii) sodium bound. The RMSD was calculated following the alignment of protein …
Figure 6—figure supplement 2
Sodium binding during simulations.
Distance of closest sodium ion to the Na1 binding site (measured as the centre of mass of Cα atoms of residues making the Na1 binding site – S114, N115, S128, T132, and E260) throughout simulations …
Figure 7 with 2 supplements
Schematic of mechanism.
Pantoate binding to the cross-over region between TM4b and TM9b of the substrate-free structure (A) elicits a conformational change in TM4b (red arrow) (B). The change in conformation of the core …
Figure 7—figure supplement 1
Pantoate binding to an outward-facing state model.
Pantoate binding region of ASBTNM(Pan) and panel domain of ASBTNM(TCH) superposed separately on the core and panel regions of the outward-facing structure of ASBTYF (4N7X; pink). Pantoate would …
Figure 7—video 1
Morph between ASBTNM(Pan) and ASBTNM(ns) focussed on TM4b.
The colouring is shown as in Figure 1. The sodium ions and pantoate from the ASBTNM(Pan) structure are held rigid during the morph of the protein atoms.
Author response image 1
The 2mFo-DFc density is at 1σ, the mFo-DFc density is at 2.
5σ.
Tables
Table 1
Data processing and refinement statistics.
| ASBTNM(Pan) | ASBTNM(ns) | |
|---|---|---|
| Wavelength (Å) | 0.9999 | 0.9999 |
| Resolution range | 43.78–2.3 (2.382–2.3) | 58.96–2.1 (2.175–2.1)* |
| Space group | C2 | P 21 21 21 |
| Unit cell: a, b, c (Å),α, β, γ (°) | 85.0 89.4 53.1 90 124.4 90 | 49.5 80.6 86.5 90 90 90 |
| Total reflections | 55,444 (3034) | 146,457 (14,741) |
| Unique reflections | 14,273 (1201) | 20,744 (2032) |
| Multiplicity | 3.9 (2.5) | 7.1 (7.3) |
| Completeness (%) | 97.7 (82.8) | 99.4 (99.3) |
| Mean I/sigma(I) | 5.7 (1.7) | 6.7 (2.0) |
| Wilson B-factor | 36 | 30 |
| R-merge | 0.1181 (0.4632) | 0.1356 (0.8541) |
| R-meas | 0.1368 (0.5779) | 0.1467 (0.9198) |
| R-pim | 0.06752 (0.3397) | 0.05402 (0.3316) |
| CC1/2 | 0.988 (0.804) | 0.957 (0.658) |
| CC* | 0.997 (0.944) | 0.989 (0.891) |
| Reflections used in refinement | 14,259 (1201) | 20,691 (2027) |
| Reflections used for R-free | 774 (55) | 1089 (96) |
| R-work | 0.2284 (0.3488) | 0.2115 (0.2933) |
| R-free | 0.2648 (0.4564) | 0.2387 (0.2885) |
| CC(work) | 0.937 (0.861) | 0.929 (0.846) |
| CC(free) | 0.927 (0.678) | 0.835 (0.907) |
| Number of non-hydrogen atoms | 2324 | 2412 |
| Macromolecules | 2276 | 2282 |
| Ligands | 34 | 135 |
| Solvent | 25 | 34 |
| Protein residues | 310 | 310 |
| RMS (bonds) | 0.002 | 0.002 |
| RMS (angles) | 0.44 | 0.53 |
| Ramachandran favoured (%) | 98.05 | 99.03 |
| Ramachandran allowed (%) | 1.95 | 0.97 |
| Ramachandran outliers (%) | 0 | 0 |
| Rotamer outliers (%) | 0.85 | 0.84 |
| Clashscore | 2.52 | 3.25 |
| Average B-factor | 52.2 | 41.5 |
| Macromolecules | 52.3 | 40.3 |
| Ligands | 46.0 | 69.4 |
| Solvent | 47.6 | 42.8 |
| Number of TLS groups | 1 | 1 |
-
*
Statistics for the highest resolution shell are shown in parentheses.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Nesseria meningitidis) | ASBTNM | https://doi.org/10.1038/nature10450 | ASBTNM | |
| Recombinant DNA reagent | PWaldo GFPd-3C (plasmid) | Hatton et al., 2022 | Modified from original PWaldo GFPd vector https://doi.org/10.1110/ps.051466205 | |
| Strain, strain background (Escherichia coli) | Lemo21(DE3) | New England Biolabs | ||
| Chemical compound | Pantoate | Merck Life Science UK | (R)-Pantoic acid sodium salt |
