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. 2012 Apr 27;287(18):14994-5000.
doi: 10.1074/jbc.M112.340281. Epub 2012 Mar 8.

Dissociation of ATP-binding Cassette Nucleotide-Binding Domain Dimers Into Monomers During the Hydrolysis Cycle

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Free PMC article

Dissociation of ATP-binding Cassette Nucleotide-Binding Domain Dimers Into Monomers During the Hydrolysis Cycle

Maria E Zoghbi et al. J Biol Chem. .
Free PMC article

Abstract

ATP-binding cassette (ABC) proteins have two nucleotide-binding domains (NBDs) that work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. In particular, it is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or opening of the dimers, with the NBDs remaining in contact during the hydrolysis cycle. We studied a prototypical ABC NBD, the Methanococcus jannaschii MJ0796, using spectroscopic techniques. We show that fluorescence from a tryptophan positioned at the dimer interface and luminescence resonance energy transfer between probes reacted with single-cysteine mutants can be used to follow NBD association/dissociation in real time. The intermonomer distances calculated from luminescence resonance energy transfer data indicate that the NBDs separate completely following ATP hydrolysis, instead of opening. The results support ABC protein NBD association/dissociation, as opposed to constant-contact models.

Figures

FIGURE 1.
FIGURE 1.
Structure and function of NBDs. A, structure of nucleotide-bound MJI-C14. Each monomer is represented in a different yellow tone. ADP and Pi are shown in space-filling representation (gray), and Cys-14, Gln-171, and Trp-174 are shown in sticks representation (cyan, magenta, and red, respectively). This structure is based on the MJ0796-E171Q/G174W coordinates (Protein Data Bank (PDB) 3TIF). B, emission spectra from ATP-bound MJ-C14 dimers labeled with Tb3+ only (Tb only, black), fluorescein only (F only, blue), or Tb3+ and fluorescein (Tb/F, red). The emission spectrum of MJ-CL subjected to the Tb3+/fluorescein labeling procedure is also shown (MJ-CL Tb/F, green trace). The inset is a zoomed view showing the donor only (black) and donor-acceptor traces (red). Protein concentration was 2 μm, and 2 mm ATP was added 10 min before collecting the spectra. Intensities were normalized to the 546-nm MJ-C14 Tb/F Tb3+ peak, except for the Tb3+ only trace, which was scaled to the Tb3+ 585-nm peak of MJ-C14 Tb/F. C, typical MJ-C14 LRET intensity changes in response to increasing ATP concentration. The sensitized fluorescein emission was measured at ATP concentrations ranging from zero (bottom trace) to 500 μm (top trace). Intermediate ATP concentrations were 5, 10, 15, 20, 30, 40 (red), 50, 100, 200, and 500 μm. Traces correspond to data normalized to the peak emission in 500 μm ATP. The spectra were acquired at 5-min intervals between sequential ATP additions. The solutions were nominally divalent cation-free and contained 1 mm EDTA, to prevent ATP hydrolysis. D, summary of the dependence of the LRET signal on ATP concentration. The sensitized emission data were obtained from experiments similar to those in panel C and are shown as means ± S.E. (n = 5 for each protein). S.E. values smaller than the symbol size are not shown.
FIGURE 2.
FIGURE 2.
Time course of NBD association/dissociation monitored by LRET and Trp quenching. A, changes in MJ-C14 Trp fluorescence (black) and fluorescein sensitized emission (red) in response to ATP and Mg-ATP. MJ-C14 used to detect Trp fluorescence was not labeled with LRET probes. ATP (2 mm) was added at the first arrow, and MgCl2 (10 mm) was added at the second arrow. Traces show signals normalized to the total change. B, changes in MJI-C14 Trp fluorescence (black) and fluorescein sensitized emission (red) in response to ATP and Mg-ATP. See panel A for details. C, response of MJ-C14 (black) and MJI-C14 (green) LRET signal to low Mg-ATP concentration. Mg2+ at a concentration of 10 mm was present from the beginning. For all panels, the protein concentration was 2 μm, and the traces are representative of data from >5 similar experiments.
FIGURE 3.
FIGURE 3.
Emission decays of donor- and donor-acceptor-labeled NBDs. A, effect of ATP on sensitized fluorescein emission decay from MJ-C14. Black trace (Tb only), emission of MJ-C14 labeled with Tb3+ only, measured at 490 nm. Blue (No ATP) and red traces (ATP), sensitized fluorescein emission of MJ-C14 labeled with Tb3+ and fluorescein, measured at 520 nm in the absence or presence of 2 mm ATP, respectively. Green trace (MJ-CL), sensitized fluorescein emission (measured at 520 nm) of Cys-less protein (MJ-CL) subjected to the Tb3+ and fluorescein labeling procedure. When present, ATP concentration was 2 mm. Tb3+ only and ATP intensities were normalized to their corresponding values at 1,000 μs, and the No ATP and MJ-CL intensities were normalized to the ATP intensity at 1,000 μs. The traces are representative of 5 similar experiments. B, effect of ATP on sensitized Cy3 emission decay from MJ-C14. Details are as in panel A, but Cy3 was used as acceptor instead of fluorescein, with the sensitized emission measured at 570 nm. C, effect of Mg-ATP on the sensitized fluorescein emission decay of Tb3+/fluorescein-labeled MJ-C14. Semilog plot of the sensitized fluorescein decay measured in 2 mm ATP (red) and after the addition of MgCl2 (green). The decay in the absence ATP (recorded in the same sample at the beginning of the experiment) was subtracted from the ATP and Mg-ATP decays, resulting in traces that can be fit to a single exponential function (black lines over the ATP and Mg-ATP traces). The decay from MJ-C14 labeled with Tb3+ alone (black) is also shown. ATP and Tb3+ only were normalized to their intensities at 1,000 μs, whereas the Mg-ATP data were normalized to the ATP intensity at 1,000 μs. D, effect of Mg-ATP on the sensitized Cy3 emission decay of Tb3+/Cy3-labeled MJ-C14. Details are as in panel C, but Cy3 was used as acceptor instead of fluorescein.

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