Exploring conformational equilibria of a heterodimeric ABC transporter
- Ruhr-Universität Bochum, Germany
- Freie Universität Berlin, Germany
- University of Zurich, Switzerland
- ETH Zurich, Switzerland
Figures
Figure 1 with 2 supplements
Labeling sites and putative conformational switch.
Schematic of spin-labeled sites in the extracellular, intracellular and NBD regions of TM287/288 in (A) the inward-facing apo crystal structure (PDB: 4Q4H) and in (B) the outward-facing homology …
Figure 1—figure supplement 1
Comparison between simulated and experimental distance distributions.
Simulations of the distance distribution probability for the six spin-labeled pairs in the IF (cyan) and OF (magenta) states performed with MMM2015 with the rotamer library at ambient temperature …
Figure 1—figure supplement 2
Hoechst 33342 stimulated ATPase activities of wildtype TM287/288 and spin-labeled mutants reconstituted into proteoliposomes.
Stimulation of ATPase hydrolysis of wildtype TM287/288 and six spin-labeled mutants reconstituted into proteoliposomes was determined in the absence (basal activity) and in the presence of 50 µM, …
Figure 2 with 4 supplements
DEER analysis of spin-labeled TM287/288.
Q-band background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (left) and corresponding distance distribution (right) calculated using DeerAnalysis2015 and normalized by area. …
Figure 2—figure supplement 1
Primary DEER traces of spin-labeled pairs in wildtype TM287/288.
Q-band DEER traces [V(t)/V(0)] with the fitted background using homogeneous stretched exponential decay with dimensions of 1.5–3 for the six spin-labeled pairs engineered into wildtype TM287/288. …
Figure 2—figure supplement 2
DEER analysis of TM287/288 in proteoliposomes.
Q-band DEER trace [V(t)/V(0)] with the background fit (left), background corrected DEER trace [F(t)/F(0)] with fitted distribution function (center) and the corresponding distance distribution …
Figure 2—figure supplement 3
DEER analysis of spin-labeled pairs in wildtype TM287/288 with additional nucleotide analogs and ADP-Mg.
Q-band background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (left) and corresponding distance distribution (right) for the six spin-labeled pairs engineered into wildtype …
Figure 2—figure supplement 4
DEER analysis of wildtype TM287/288 in the presence of 2.5 mM or 14 mM ATP and 2.5 mM EDTA.
Q-band DEER traces [V(t)/V(0)] (left), background corrected DEER traces [F(t)/F(0)] with fitted distribution functions (center) and corresponding distance distributions (right) for three …
Figure 3
Inhibition of ATPase activity of TM287/288 and BmrCD by vanadate and nucleotides.
Km and vmax values for ATP hydrolysis by TM287/288 at 50°C (A) and 25°C (C) and BmrCD at 25°C (E) were determined by measuring ATPase activities at increasing ATP concentrations. Inhibition of ATP …
Figure 4 with 2 supplements
DEER analysis of spin-labeled TM287/288 carrying the E-to-Q mutation.
Q-band background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (left) and corresponding distance distribution (right) calculated using DeerAnalysis2015 and normalized by area. …
Figure 4—figure supplement 1
Primary DEER traces of spin-labeled pairs in TM287/288 carrying the E-to-Q mutation.
Q-band DEER traces [V(t)/V(0)] with the fitted background using homogeneous stretched exponential decay with dimensions of 1.5–3 for the six spin-labeled pairs engineered on the E-to-Q mutant. Color …
Figure 4—figure supplement 2
DEER analysis of spin-labeled pairs in TM287/288 carrying the E-to-Q substitution with additional nucleotide analogs.
Q-band background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (left) and corresponding distance distribution (right) for the six spin-labeled pairs of TM287/288 carrying the …
Figure 5 with 1 supplement
Nucleotide ranking according to the ability to populate the OF conformation.
Three spin-labeled pairs of TM287/288 representing the extracellular region (150TM287/295TM288), intracellular region (131TM288/248TM288) and the NBDs (460TM287/363TM288) were used for the analysis …
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Figure 5—source data 1
Parameters of the two-Gaussian fit.
- https://doi.org/10.7554/eLife.20236.017
Figure 5—figure supplement 1
Two-Gaussian fit DEER analysis of three selected spin-labeled pairs of TM287/288.
Q-band background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (left) and corresponding distance distribution (right) for the three selected spin-labeled pairs engineered on …
Figure 6 with 1 supplement
DEER distance distribution of samples snap frozen from room temperature and 80°C.
Q-band DEER distance distribution for the six spin-labeled pairs engineered on wildtype TM287/288. Distance distributions normalized by area are shown for the samples incubated at room temperature …
Figure 6—figure supplement 1
DEER analysis of wildtype TM287/288 snap frozen from 80°C.
Q-band DEER traces [V(t)/V(0)], background corrected DEER traces [F(t)/F(0)] with fitted distribution function and corresponding distance distributions (from Figure 6) for the six spin-labeled pairs …
Figure 7
Reversibility of the disengagement of the NBDs at high temperatures.
Q-band primary DEER data [V(t)/V(0)] with the background fit (left), background corrected Form factor [F(t)/F(0)] with the fit (center) and corresponding distance distribution (right) for the 350TM28…
Figure 8
Nucleotide binding to the degenerate site stabilizes cross-NBD contacts of inward-facing TM287/288.
The interfaces between the NBDs were analyzed by the PISA server. (A) Analysis of AMP-PNP-Mg bound TM287/288 (PDB: 4Q4A), (B) analysis of apo TM287/288 (PDB: 4Q4A). The left panel shows a top view …
Figure 9 with 2 supplements
DEER analysis of spin-labeled pairs in the NBDs of BmrCD and MsbA.
Q-band background-corrected DEER traces [F(t)/F(0)] with fitted distribution function (left) and corresponding distance distribution (right). (A) Spin labeled pair 348BmrC/532BmrD in wildtype BmrCD …
Figure 9—figure supplement 1
DEER analysis of BmrCD and MsbA.
Q-band primary DEER traces [V(t)/V(0)] with fitted background for the spin-labeled pair 348BmrC/532BmrD in (A) wildtype BmrCD and (B) in BmrCD containing the E-to-Q mutation; (C) data for the …
Figure 9—figure supplement 2
DEER analysis of wildtype BmrCD in the presence of 2.5 and 10 mM ATP.
Q-band DEER trace [V(t)/V(0)] with the background fit (left), background corrected DEER trace [F(t)/F(0)] with fitted distribution function (center) and the corresponding distance distribution …
Figure 10
Proposed mechanism for heterodimeric ABC exporters.
Heterodimeric ABC exporters exhibit the following major conformations: IF with separated NBDs with no nucleotide bound (state 0), IF with partially contacting NBDs and one nucleotide bound to the …
Author response image 1
Tables
Table 1
ATPase activities in detergent.
Protein | Nucleotide | Temperature [°C] | ATPase activity [nmol Pi/min/mg protein] | Turnover per transporter [min−1] | % of wildtype | |
|---|---|---|---|---|---|---|
TM287/288 | wildtype | ATP - Mg | 50 | 2141 ± 67 | 284 | - |
25 | 86.1 ± 2.5 | 11.4 | - | |||
ATP – Mg +1 mM vanadate | 50 | 165 ± 20 | 21.9 | 7.71 | ||
ATP – Mg +2.5 mM EDTA | 50 | <0.1 | <0.01 | <0.005 | ||
AMP-PNP - Mg | 25 | <0.1 | <0.01 | <0.005 | ||
E517QTM288 | ATP - Mg | 25 | 0.165 ± 0.015 | 0.0219 | 0.192 | |
Spin-labeled TM287/288 | 350TM287/475TM288 | ATP - Mg | 50 | - | - | 193* |
460TM287/363TM288 | ATP - Mg | 50 | - | - | 212* | |
131TM288/248TM288 | ATP - Mg | 50 | - | - | 156* | |
231TM287/304TM287 | ATP - Mg | 50 | - | - | 146* | |
150TM287/295TM288 | ATP - Mg | 50 | - | - | 112* | |
50TM287/271TM287 | ATP - Mg | 50 | - | - | 73* | |
BmrCD | wildtype | ATP - Mg | 25 | 22.9 ± 0.5 | 3.24 | - |
E592QBmrD | ATP - Mg | 25 | 0.633 ± 0.053 | 0.0896 | 2.76 | |
MsbA | wildtype | ATP - Mg | 30 | 135 ± 9 | 18.4 | - |
561MsbA | ATP - Mg | 30 | 122 ± 17 | 16.6 | 90.4 | |
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* values are given in respect to an internal wildtype control in each measurement.
Table 2
Ki determination.
Protein | Temperature [°C] | Km for ATP [µm]∗ | Competitor | IC50 [µm]∗ | Ki [µm]† |
|---|---|---|---|---|---|
TM287/288 | 50 | 7.78 ± 1.10 | AMP-PNP | 16.1 ± 1.6 | 0.246 ± 0.042 |
ATPγS | 2.86 ± 0.08 | 0.0439 ± 0.0062 | |||
ADP | 681 ± 160 | 10.4 ± 2.9 | |||
Vanadate | 4.90 ± 0.29 | 0.0751 ± 0.0114 | |||
25 | 3.27 ± 0.42 | AMP-PNP | 179 ± 75 | 1.17 ± 0.51 | |
ATPγS | 31.4 ± 2.8 | 0.204 ± 0.032 | |||
ADP | 505 ± 21 | 3.28 ± 0.44 | |||
BmrCD | 25 | 343 ± 24 | AMP-PNP | 174 ± 24 | 21.0 ± 3.2 |
ATPγS | 221 ± 24 | 26.7 ± 3.3 |
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∗ Km and IC50 values and standard errors were obtained from fits shown in Figure 3.
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† Ki values and standard errors were calculated based on the given Km and IC50 values as described in the materials and methods.
