Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 4;286(9):7104-15.
doi: 10.1074/jbc.M110.201178. Epub 2010 Dec 29.

Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus

Ariane Zutz  1 , Jan Hoffmann, Ute A Hellmich, Clemens Glaubitz, Bernd Ludwig, Bernd Brutschy, Robert Tampé
Affiliations

Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport complex TmrAB from Thermus thermophilus

Ariane Zutz et al. J Biol Chem. .

Abstract

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a K(m) of 0.9 mm and k(cat) of 9 s(-1) at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Expression and purification of the TmrAB complex. A, topology model of the ABC transporters TmrA and TmrB. Each polypeptide is composed of a TMD and a NBD. Both TMDs consist of six transmembrane helices as predicted by HMMTOP 2.0 (32). Conserved ABC motifs (Walker A/B and C-loop) are found in the NBDs of both subunits. The position of the putative catalytic residue next to the Walker B motif is indicated. In TmrB, the catalytic glutamate is substituted by an aspartate. At the C terminus of TmrA, a TEV cleavage site followed by a His10 tag was introduced for purification of the ABC transport complex. B, expression of TmrA and TmrB. After isopropyl β-d-thiogalactopyranoside induction, the expression of TmrAB in E. coli BL21 (DE3) was analyzed by SDS-PAGE (10%, Coomassie staining, upper panel) and immunoblotting using the anti-His antibody (lower panel). 5 μg of total protein were applied per lane. C, purification of the TmrAB complex. Wild type and various mutants (E523Q/WT and WT/D500N) were solubilized in 1% DDM and purified via metal affinity chromatography. The heterodimeric ABC transport complex was analyzed by SDS-PAGE (10%, Coomassie staining).
FIGURE 2.
FIGURE 2.
TmrAB forms a stable heterodimeric complex. A, MALDI-TOF-MS of purified TmrAB recorded in the linear positive ion mode. Peak A and B have masses of 69.1 and 64.4 kDa, corresponding to TmrA after TEV treatment and TmrB, respectively. Single and multiple charged states are indicated (sum of 100 laser shots). B, gel filtration of purified TmrAB. Purified TmrAB was separated on a TSK gel 3000SW column in the presence of 0.02% DDM (w/v). TmrAB elutes at an apparent mass of approximately 250 kDa. The void volume of the column is indicated with V0, and the retention volume is indicated with Vt. C, LILBID-MS anion spectra of TmrAB after TEV cleavage recorded at low laser (upper panel) or high laser intensity (lower panel). At low laser intensity the charge distribution corresponds to a mass of 148 kDa indicated by the dotted lines. The respective charge states are labeled. At high laser intensity, an additional charge distribution, corresponding to a mass of 133 kDa, is visible. The charge distribution and the corresponding charge states are labeled.
FIGURE 3.
FIGURE 3.
Hoechst 33342 uptake by TmrAB in inside-out vesicles. A, orientation of wild type and mutant TmrAB in IOVs. IOVs were prepared from wild type and E523Q/D500N TmrAB expressing E. coli cells and incubated overnight at 30 °C in the absence or presence of TEV protease. As a positive control, IOVs were solubilized with 2% Triton X-100 (TX-100) before TEV cleavage. 20 μg of total protein were separated by SDS-PAGE and orientation of TmrAB was determined via immunoblotting using an anti-His antibody. B, wild type TmrAB mediates Hoechst 33342 uptake in IOVs. IOVs derived from E. coli cells expressing wild type TmrAB (black line), E523Q/D500N TmrAB (green line), and the empty pET22b+ vector (cyan line), respectively, were incubated with 0.75 μm of Hoechst 33342. Membrane translocation was initiated after 2 min by the addition of MgATP (1.5 mm). The fluorescence was monitored over time at 50 °C with excitation and emission wavelengths of 355 and 457 nm, respectively. As control, Hoechst 33342 uptake was measured at 37 °C using IOVs containing wild type TmrAB (gray line). C, inhibition of Hoechst 33342 uptake by NaN3 and nigericin. The experiments were performed as described in C using IOVs containing wild type TmrAB (black line). The reaction was carried out in the presence of 10 mm NaN3 (gray line) to inhibit the F1F0-ATPase. To dissipate proton motive force, 1 μm of nigericin was added after 5 min (blue line). D, inhibition of Hoechst 33342 uptake by verapamil. Hoechst 33342 uptake was carried out in wild type IOVs as described above. The reaction was inhibited by titration of verapamil, resulting in an IC50 value of 83 μm.
FIGURE 4.
FIGURE 4.
ATP hydrolysis of purified TmrAB. A, temperature dependence of the ATPase of TmrAB. The basal ATPase activity of DDM-solubilized wild type TmrAB (60 nm) was analyzed by monitoring the release of inorganic phosphate at different temperatures. The reaction was carried out for 5 min in the presence of 5 mm ATP. B, inhibition of ATPase activity. The ATPase of wild type TmrAB (60 nm) was analyzed for 4 min at 68 °C in the presence of BeFx (5 mm), vanadate (5 mm), and NaN3 (10 mm). C, Hoechst 33342 inhibits ATP hydrolysis by TmrAB. ATP hydrolysis of purified TmrAB (WT) was carried out as described in B in the presence of increasing concentrations of Hoechst 33342. ATPase activity was inhibited in a dose-dependent manner resulting in an IC50 value of 95 μm. D, determination of Km,ATP and kcat values. The ATPase activity of TmrAB (60 nm) was measured as a function of ATP concentration for 4 min at 68 °C. Wild type TmrAB (filled circles) was ATPase active, resulting in a kcat of 8.8 s−1 and a Km,ATP of 0.92 mm. The E523Q/D500N mutant (filled triangles) shows no detectable ATPase activity above background.
FIGURE 5.
FIGURE 5.
Nucleotide trapping by wild type and E523Q/WT TmrAB complex. A, binding of 8-azido-[α-32P]ATP and 8-azido-[γ-32P]ATP by wild type and E523Q/WT TmrAB. Purified wild type and E523Q/WT complexes (0.5 μm of each) were incubated with radiolabeled 8-azido-ATP (4 μm) for 4 min at room temperature in the presence or absence of an excess of cold ATP (5 mm). Additionally, 0.4 mm orthovanadate (Vi) was added to the wild type complex. Photo-cross-linking was performed for 4 min on ice. The proteins (0.6 μg/lane) were separated by SDS-PAGE (10%), and the photo-cross-linked proteins were visualized by autoradiography. B, 8-azido-ATP photo-cross-linking under hydrolysis conditions. Purified wild type and E523Q/WT mutant TmrAB (0.5 μm) were incubated at 68 °C, followed by the addition of either 8-N3-[α-32P]ATP or 8-N3-[γ-32P]ATP. The wild type complex was supplemented with 0.4 mm vanadate (Vi) to trap nucleotides in the posthydrolysis state. Cold ATP (5 mm) was used to probe for specific cross-linking. After cooling down, the samples were incubated with an excess of cold ATP to demonstrate tight occlusion of nucleotides. Subsequently, the cross-link reaction was performed for 4 min on ice. As a control, binding of labeled azido-ATP at room temperature (RT) is shown for the wild type complex. Nucleotide binding was analyzed as described above.
FIGURE 6.
FIGURE 6.
Stoichiometry of bound nucleotides in the pre- and posthydrolysis state. A, nucleotide trapping in TmrAB is temperature-dependent. Wild type TmrAB was incubated in the presence of ATP (0.7 mm) and 5 mm orthovanadate (Vi) for 3.5 min at 4 and 68 °C, respectively. Tracer amounts of [α-32P]ATP were added during reaction. Prior to spin-down, the samples were supplemented with an excess of cold ATP to verify stable trapping of nucleotides. After separation by rapid gel filtration, the amount of bound nucleotides was determined by β-counting. B, vanadate-dependent nucleotide trapping under hydrolysis conditions. Trapping experiments with wild type TmrAB were performed for 3.5 min at 68 °C with ATP (0.7 mm) using tracer amounts of [α-32P]ATP (black bars) and [γ-32P]ATP (white bars), respectively. Protein samples were incubated in the absence or presence of 5 mm orthovanadate (Vi). As competitor, either ATP or AMP (10 mm of each) was added. After cooling down to room temperature, the samples were supplemented with 2 mm ATP to demonstrate stable occlusion of nucleotides. Free ATP was removed via rapid gel filtration (spin-down). The amount of trapped nucleotides was determined as described above. C, effects of mutations on nucleotide binding and hydrolysis. Trapping of [α-32P]ATP (black bars) and [γ-32P]ATP (white bars) by wild type and mutant TmrAB was analyzed as described in B. Nucleotide trapping of the ATPase-deficient E523Q/WT, E523Q/D500N, and E523Q/D500E mutants was performed in the absence of vanadate. The calculated stoichiometries result from at least three independent experiments. D, determination of nucleotide composition by TLC. Trapped nucleotides were analyzed by thin layer chromatography. The amount of either [α-32P]ATP or [α-32P]ADP was quantified via phosphorus imaging, and the amounts of ATP (black bars) and ADP (white bars) were calculated for wild type and mutant TmrAB. The data are represented as the means ± S.E. of at least three independent experiments. E, model of ATP hydrolysis in the TmrAB complex. Vanadate traps ATP in WT TmrAB, forming a posthydrolysis state, with one ADP-Vi bound in site I and one ATP bound in site II. Mutation of the catalytic base residue Glu-523 mediates the vanadate-independent occlusion of two ATP in the prehydrolysis state. Introduction of a new catalytic base residue (Asp-500) in TmrB leads to an ATPase active site II (slow hydrolysis), which depends on formation of the posthydrolysis state in site I. The exact data are shown in C and D. The vanadate-trapped posthydrolysis state is indicated in red. Mutations of the putative catalytic base residues are labeled in orange.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Higgins C. F. (1992) Annu. Rev. Cell Biol. 8, 67–113 - PubMed
    1. Dean M., Rzhetsky A., Allikmets R. (2001) Genome Res. 11, 1156–1166 - PubMed
    1. Locher K. P. (2009) Philos Trans. R. Soc. Lond B Biol. Sci. 364, 239–245 - PMC - PubMed
    1. Davidson A. L., Dassa E., Orelle C., Chen J. (2008) Microbiol. Mol. Biol. Rev. 72, 317–364 - PMC - PubMed
    1. Lubelski J., Konings W. N., Driessen A. J. (2007) Microbiol. Mol. Biol. Rev. 71, 463–476 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources