ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter
- Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Germany
Figures
Figure 1 with 3 supplements
Accessible-volume (AV) simulations of LD fluorophores on TmrAB variants.
AV simulations were performed for LD555 (donor) and LD655 (acceptor) fluorophores attached to the selected TmrAB labeling sites to assess whether donor–acceptor distances are suitable for single-molecule Förster resonance energy transfer (smFRET) measurements (Kalinin et al., 2012). (a) TmrABNBD (TmrAC416BL458C) and (b) TmrABPG (TmrAC416A, T61CBR56C) in the inward-facing wide (IFwide; PDB: 6RAN, left) and outward-facing open (OFopen; PDB: 6RAH, right) conformations. The approximate membrane position is indicated by the dashed gray line. TmrA is shown in blue with LD655 (orange) and TmrB in yellow with LD555 (green), with labeling positions indicated (416/458 for TmrABNBD; 61/56 for TmrABPG). For illustration, fluorophores are shown in a defined configuration; however, in experiments, donor and acceptor dyes are stochastically attached, resulting in random distribution between subunits.
-
Figure 1—source data 1
Excel file containing the data for the accessible-volumen (AV) simulation for Figure 1.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Quality of TmrAB purification and fluorophore labeling.
(a) SDS-PAGE analysis (10%, reducing gel, Coomassie staining) of successive purification steps: M, molecular weight marker; L, cell lysate; B, Ni-NTA beads after incubation with lysate; FT, flow-through; W, wash; E, eluted TmrAB; BE, buffer-exchanged sample; C, concentrated protein; CFT, concentrator flow-through; 1 and 2, first and second peaks eluted from size-exclusion chromatography (SEC). Only the second peak was used for subsequent Förster resonance energy transfer (FRET) experiments. (b) SEC (Superdex 200 increase 10/300 GL) showing monodisperse labeled TmrAB and efficient removal of free fluorophores. A representative chromatogram of TmrABPG is shown. (c) ATP hydrolysis activity of purified wild-type TmrAB (60 nM TmrABwt) measured at 40°C for 7 min using the Malachite Green assay. Released inorganic phosphate (Pi) was quantified and data fitted to Michaelis–Menten kinetics, yielding Km = 0.97 ± 0.28 mM and kcat = 2.57 ± 0.38 s–1. (d–f) Analytical SEC (Superdex 200 increase 3.2/300) used to determine fluorophore labeling efficiencies of (d) TmrABNBD, (e) TmrABPG, and (f) TmrABPG_EQ. LD555 and LD655 labeling efficiencies were ~55% and ~53% (TmrABNBD), ~43% and ~52% (TmrABPG), and ~42% and ~52% (TmrABPG_EQ), respectively. Total cysteine occupancy exceeds 90% in all variants, reflecting near-complete dual-labeling with equimolar fluorophores.
-
Figure 1—figure supplement 1—source data 1
Excel file containing the data for Figure 1–figure supplement 1.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-figsupp1-data1-v1.xlsx
-
Figure 1—figure supplement 1—source data 2
Orignal SDS-PAGE image.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-figsupp1-data2-v1.zip
-
Figure 1—figure supplement 1—source data 3
Orignal SDS-PAGE image, labeled.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-figsupp1-data3-v1.zip
Figure 1—figure supplement 2
Förster resonance energy transfer (FRET) properties of labeled TmrAB variants.
(a) Time-correlated single-photon counting histograms of LD555 (left) and LD655 (middle) measured for free dye in buffer (black), LD555/LD655-labeled TmrABNBD (orange), and LD555/LD655-labeled TmrABPG (blue). Amplitude-weighted average fluorescence lifetimes (τ) are summarized in the histogram (right), confirming sufficient fluorophore mobility for reliable FRET measurements. (b–d) Donor-excited ensemble emission spectra (550–700 nm, excitation 520 nm) of stochastic LD555/LD655-labeled (b) TmrABNBD, (c) TmrABPG, and (d) the slow-turnover variant TmrABPG_EQ measured at increasing ATP concentrations. Spectra were normalized to donor intensity in the apo state. ATP-dependent donor quenching and acceptor sensitization demonstrate that all variants retain FRET capability. (e–g) Fractional fluorescence changes, (F-F0)/F0, plotted as a function of ATP concentration for (e) TmrABNBD, (f) TmrABPG, and (g) TmrABPG_EQ, where F is the acceptor emission intensity and F0 the intensity in the apo state. Data were fitted with a hyperbolic (Langmuir-type) binding model to determine apparent Kd,ATP values, which are consistent with ensemble FRET measurements of ATP binding.
-
Figure 1—figure supplement 2—source data 1
Excel file containing the source data 1 for Figure 1–figure supplement 2.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-figsupp2-data1-v1.xlsx
-
Figure 1—figure supplement 2—source data 2
Excel file containing the source data 2 for Figure 1–figure supplement 2.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-figsupp2-data2-v1.xlsx
Figure 1—figure supplement 3
Effect of fluorophore labeling and nanobody binding on TmrAB transport activity.
Single-liposome transport assays were performed by flow cytometry using mean fluorescence intensity at 527 nm (MFI527) as a readout of peptide transport. (a) TmrAB (0.6 µM) reconstituted into liposomes (~50 transporters per liposome) was incubated with C4F peptide (30 µM; RRYCFKSTEL, F, fluorescein) in the presence of 3 mM ATP (green) or 3 mM ADP (orange) at 40°C for 5 min. After extensive washing, proteoliposomes were analyzed by flow cytometry. (b) Flow cytometry gating strategy. Forward scatter area (FSC-A) versus side scatter area (SSC-A; left) was used to identify homogeneous proteoliposomes, forward scatter height (FSC-H) versus FSC-A (middle) to select singlets, and fluorescence intensity at 527 nm (F527; right) to determine the mean fluorescence intensity (MFI) values of the gated population. (c) Transport activity of unlabeled wild-type TmrAB (TmrABWT) compared with LD555/LD655-labeled TmrABPG. (d) Effect of nanobody binding on TmrABwt transport activity. Assays were performed in the absence or presence of Nb9F10S63C at a 1:1 molar ratio (0.6 µM each). Fluorophore labeling at the periplasmic gate and nanobody binding did not measurably affect TmrAB transport activity.
-
Figure 1—figure supplement 3—source data 1
Excel file containing the source data 1 for Figure 1–figure supplement 3.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig1-figsupp3-data1-v1.xlsx
Figure 2 with 2 supplements
ATP-induced conformational changes of TmrAB analyzed by single-molecule Förster resonance energy transfer (smFRET).
(a) Experimental setup. TmrABNBD (left) and TmrABPG (right) were labeled with LD555/LD655 and immobilized on PEGylated coverslips via a biotinylated conformation-independent, TmrB-specific nanobody (Nb9F10S63C) (Hofmann et al., 2019; Nocker et al., 2026). (b) smFRET imaging was performed using total internal reflection fluorescence (TIRF) microscopy with alternating laser excitation (ALEX; donor: 532 nm; acceptor: 640 nm). Emission was collected in donor (498–620 nm) and acceptor (662–710 nm) channels. Donor emission upon donor excitation (), sensitized acceptor emission upon donor excitation (), and direct acceptor emission upon acceptor excitation () were used to calculate FRET efficiency (E) and stoichiometry (S). Data were analyzed using DeepFRET (Thomsen et al., 2020). (c) Representative TmrABNBD trace in ATP-bound state (top) and corresponding E/S plot (bottom). Donor emission upon donor excitation is shown in green (photon counts per frame), acceptor emission upon donor excitation in orange, FRET efficiency (E) in black, and stoichiometry (S) in gray. The initial increase in donor fluorescence reflects photophysical equilibration and instrumental stabilization and does not affect E or S, which are ratio-based and time-invariant (Hellenkamp et al., 2018). (d, e) Population analysis. FRET efficiency (E) histograms for (d) TmrABNBD and (e) TmrABPG are shown for apo (top) and ATP-bound (bottom; 3 mM ATP) conditions. Histograms were fitted with two Gaussian populations corresponding to the apo state (blue; defined from apo measurements) and ATP-bound state (orange; defined from saturating ATP fits). Dotted vertical lines indicate mean E values of each population, and relative fractions (Gaussian areas) are summarized schematically in each panel.
-
Figure 2—source data 1
Excel file containing the source data 1 for Figure 2.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Representative single-molecule Förster resonance energy transfer (smFRET) traces of TmrABNBD.
Representative smFRET traces of TmrABNBD recorded (a) in the absence of ATP and (b, c) in the presence of 3 mM ATP. Hidden Markov modeling (HMM) classified ATP-bound traces as (b) static or (c) dynamic based on the absence or presence of transitions between ATP-free and ATP-bound conformational states. Donor fluorescence upon donor excitation is shown in green, acceptor fluorescence upon donor excitation in orange, FRET efficiency (E) in black, and stoichiometry (S) in gray.
-
Figure 2—figure supplement 1—source data 1
Excel file containing the source data 1 for Figure 2–figure supplement 1.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig2-figsupp1-data1-v1.xlsx
-
Figure 2—figure supplement 1—source data 2
Excel file containing the source data 2 for Figure 2–figure supplement 1.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig2-figsupp1-data2-v1.xlsx
Figure 2—figure supplement 2
Representative single-molecule Förster resonance energy transfer (smFRET) traces of TmrABPG.
Representative smFRET traces of TmrABPG recorded (a) in the absence of ATP and (b, c) in the presence of 3 mM ATP. Hidden Markov modeling (HMM) classified ATP-bound traces as (b) static or (c) dynamic based on the absence or presence of transitions between ATP-free and ATP-bound conformational states. Donor fluorescence upon donor excitation is shown in green, acceptor fluorescence upon donor excitation in orange, FRET efficiency (E) in black, and stoichiometry (S) in gray.
Figure 3
ATP-dependent shifts in single-molecule Förster resonance energy transfer (smFRET) populations of TmrAB.
(a, c) Increasing ATP concentrations (0—3 mM) progressively shifted the population between apo and ATP-bound conformations for (a) TmrABNBD and (c) TmrABPG. FRET efficiency (E) histograms were fitted with two Gaussian populations corresponding to the ATP-free state (blue; defined from apo samples) and the ATP-bound state (orange; determined from saturating ATP conditions). Dotted vertical lines indicate the mean E values of each state, and relative fractions (Gaussian areas) are summarized schematically in each panel. (b, d) ATP-binding curves obtained by plotting the fraction of molecules in the ATP-bound states as a function of ATP concentration for (b) TmrABNBD (reporting NBD dimerization) and (d) TmrABPG (reporting periplasmic gate [PG] opening). Data were fitted with a Langmuir isotherm to determine the apparent dissociation constant Kd, ATP of each variant.
-
Figure 3—source data 1
Excel file containing the source data for Figure 3.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig3-data1-v1.xlsx
Figure 4
Identification of the outward-facing open (OFopen) conformation.
(a–c) Three complementary approaches were used to resolve the OFopen state: (a) the slow-turnover variant TmrABPG_EQ, (b) imaging in Mg2+-free buffer supplemented with EDTA, and (c) stabilizing via trans-inhibition using high concentrations of peptide substrate R9L (0.3–2 mM). Förster resonance energy transfer (FRET) efficiency (E) histograms were fitted with three Gaussian populations corresponding to the ATP-free state (blue), the ATP-bound state (orange), and the OFopen state (green). All three strategies reveal a distinct OFopen population. Dotted vertical lines indicate the mean E values of each state, and relative fractions (Gaussian areas) are summarized schematically in each panel. (d) Comparison of inter-residue distances. Distances (Å) between selected residues on the nucleotide-binding domains (NBDs) and periplasmic gate (PG) of TmrAB (Cβ–Cβ) were determined using small-molecule FRET (smFRET) (this study, detergent-solubilized TmrAB), cryogenic electron microscopy (cryo-EM) structures of nanodisc-reconstituted TmrAB (PDB 6RAH, 6RAN) (Hofmann et al., 2019); accessible-volume (AV) simulations (this study), and pulsed electron–electron double resonance (PELDOR/DEER) measurements of detergent-solubilized TmrAB (Barth et al., 2018).
-
Figure 4—source data 1
Excel file containing the source data for Figure 4.
- https://cdn.elifesciences.org/articles/110967/elife-110967-fig4-data1-v1.xlsx
Figure 5
Conformational state distribution and catalytic cycle of TmrAB under active turnover.
Schematic of the TmrAB transport cycle summarizing major conformational states and their estimated population distributions under physiological ATP concentrations (3 mM, 40°C). (a) The inward-facing apo state (IFnarrow and IFwide; blue arc) accounts for ~20% of molecules and is characterized by separated nucleotide-binding domains (NBDs) and a cytosol-accessible substrate-binding cavity. Substrate binding stabilizes the IFwide conformation (Hofmann et al., 2019). ATP binding induces NBD dimerization and formation of the ATP-bound ensemble. (b, c) Under substrate-bound turnover conditions, TmrAB proceeds via (b) OFoccluded and (c) OFopen states in which substrate release occurs. In steady-state turnover, the ATP-bound ensemble rapidly interconverts between OFoccluded and OFopen, accounting for ~25% of the ATP-bound population (green circle). These transitions occur faster than the ~200 ms temporal resolution of single-molecule Förster resonance energy transfer (smFRET) measurements, resulting in an averaged signal under turnover conditions. OFoccluded is proposed as an obligate intermediate between IF and OFopen, preventing substrate backflow by maintaining an occluded binding cavity during rearrangements of the periplasmic gate (PG) and NBDs. Although a substrate-bound OFoccluded state has not been directly observed for TmrAB, its existence is supported by structures of the homodimeric type IV transporter BmrA (Chaptal et al., 2022). Reduced ATP hydrolysis or substrate trans-inhibition enables trapping of the OFopen state. (d, e) ATP hydrolysis and phosphate (Pi) release generate post-hydrolysis return states (d) URasym and (e) URasym*. Subsequent ADP release restores the apo IF conformation, completing the transport cycle. Overall, the ATP-bound phase (b–e) represents ~55% occupancy (orange arc) with an estimated dwell time of ~310 ms, whereas the apo/ATP-rebinding phase (a) lasts ~90 ms, yielding a total cycle time of ~400 ms (kcat = 2.57 s–1). TmrA is shown in blue, TmrB in yellow, substrate as a green diamond, and nucleotides as orange symbols. Dotted gray boxes indicate the approximate position of the NBD dimer interface.
Tables
Table 1
Accessible-volume (AV) simulations of LD fluorophores on TmrAB variants.
AV simulations were performed for LD555 (donor) and LD655 (acceptor) fluorophores attached to the selected TmrAB labeling sites to assess whether donor–acceptor distances are suitable for single-molecule Förster resonance energy transfer (smFRET) measurements (Kalinin et al., 2012). AV simulations confirm that donor–acceptor distances (RDA) remain within the FRET-sensitive range in both conformations, predicting measurable changes in simulated FRET efficiencies (Esim). Cryogenic electron microscopy (cryo-EM) structures of TmrAB reconstituted in lipid nanodiscs (Hofmann et al., 2019) were used as templates, including apo and ATP-bound (3 mM ATP) states. Outward-facing open (OFopen) and outward-facing occluded (OFoccluded) structures were obtained via orthovanadate trapping (Vi) or the slow-turnover mutant TmrAE523QB (EQ).
| TmrABNBD | TmrABPG | |||||
|---|---|---|---|---|---|---|
| Conformation | PDB ID | RDA (Å) | Esim | RDA (Å) | Esim | |
| IFnarrow | (turnover) | 6RAF | 58.6 | 0.60 | 33.0 | 0.95 |
| IFwide | (turnover) | 6RAG | 57.2 | 0.63 | 30.2 | 0.96 |
| IFwide | (apo) | 6RAN | 57.8 | 0.62 | 30.3 | 0.96 |
| OFopen | (Vi) | 6RAJ | 44.8 | 0.84 | 55.8 | 0.66 |
| OFopen | (EQ) | 6RAH | 44.9 | 0.84 | 51.8 | 0.72 |
| OFoccluded | (Vi) | 6RAK | 45.4 | 0.83 | 27.0 | 0.98 |
| OFoccluded | (EQ) | 6RAI | 45.5 | 0.83 | 32.4 | 0.95 |
| URasym | (turnover) | 6RAL | 45.9 | 0.83 | 31.2 | 0.96 |
| URasym* | (turnover) | 6RAM | 47.6 | 0.80 | 31.2 | 0.96 |
-
Table 1—source data 1
Excel file containing the source data for Table 1.
- https://cdn.elifesciences.org/articles/110967/elife-110967-table1-data1-v1.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3) | Thermo Fisher Scientific | Cat. Nr.: EC0114 | Expression of His10-tagged TmrAB variants and nanobody Nb9F10S63C |
| Gene (Thermus thermophilus) | TmrABwt | Hofmann et al., 2019 | Plasmid ID: AT30 | Wild-type TmrAB |
| Gene (T. thermophilus) | TmrABNBD (TmrAC416BL458C) | Barth et al., 2020 | Plasmid ID: AT08 | TmrAB variant |
| Gene (T. thermophilus) | TmrABPG (TmrAC416A, T61CB R56C) | Barth et al., 2020 | Plasmid ID: AT27 | TmrAB variant |
| Gene (T. thermophilus) | TmrABPG_EQ (TmrAC416A, E523Q, T61CB R56C) | Barth et al., 2020 | Plasmid ID: AT01 | Slow-turnover TmrAB variant |
| Gene (Vicugna pacos) | Nb9F10S63C (nanobody) | Hofmann et al., 2019 | Plasmid ID: BH16 | TmrB-specific, conformation-independent nanobody |
| Peptide, recombinant protein | R9L | This study | N/A | RRYQKSTEL |
| Peptide, recombinant protein | C4F | This study | N/A | RRYCFKSTEL; F, fluorescein |
| Chemical compound, drug | LD555 (maleimide) | Lumidyne Technologies | CAT #4 | FRET donor |
| Chemical compound, drug | LD655 (maleimide) | Lumidyne Technologies | CAT #8 | FRET acceptor |
| Chemical compound, drug | ATP (adenosine 5'-triphosphate) | Sigma-Aldrich | CAS Nr.: 74804-12-9 | |
| Chemical compound, drug | ADP (adenosine 5'-diphosphate) | Sigma-Aldrich | CAS Nr.: 20398-34-9 | |
| Chemical compound, drug | EDTA (ethylenediaminetetraacetic acid) | Sigma-Aldrich | CAS Nr.: 6381-92-6 | |
| Chemical compound, drug | n-Dodecyl β-D-maltoside (β-DDM) | Carl Roth | CAS Nr.: 69227-93-6 | |
| Chemical compound, drug | Trolox | Sigma-Aldrich | CAS Nr.: 53188-07-1 | |
| Chemical compound, drug | Streptavidin | Sigma-Aldrich | CAS Nr.: 9013-20-1 | |
| Chemical compound, drug | Biotin-PEG11-maleimide | Sigma-Aldrich | Cat. Nr.: QBD10195 | |
| Chemical compound, drug | E. coli polar lipids | Avanti Polar Lipids | CAS Nr.: 1240502-50-4 | |
| Chemical compound, drug | Dioleoylphosphatidylcholine (DOPC) | Avanti Polar Lipids | CAS Nr.: 4235-95-4 | |
| Commercial assay, kit | Ni-NTA Agarose | Bio-Rad | Cat. Nr.: 30250 | IMAC purification of His10-tagged TmrAB |
| Software, algorithm | NanoImager software | ONI | 165527-1aaf735 | Microscope software; v1.19.16.20250510 |
| Software, algorithm | DeepFRET | Thomsen et al., 2020 | Machine-learning FRET trace classification; v2.0.5 | |
| Software, algorithm | MASH-FRET | Hadzic et al., 2018 | Hidden Markov modeling of smFRET traces; v1.3.4 | |
| Software, algorithm | FluoFit | PicoQuant | Fluorescence decay analysis; v4.6.6 (discontinued) | |
| Software, algorithm | OriginPro 2024 | OriginLab | Data analysis; v10.1.0.178 | |
| Software, algorithm | FlowJo | Waters Biosciences | RRID:SCR_008520 | Flow cytometry data analysis; v10.6.1 |
| Software, algorithm | AV simulation toolkit | Kalinin et al., 2012 | Zenodo: 3517287 | Accessible-volume simulations |
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter
eLife 15:RP110967.
https://doi.org/10.7554/eLife.110967.4
