Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain

Abstract

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.

FIGURE 1.

ATP-dependent NBD dimerization. A, Coomassie blue-stained gel of purified MJ and MJI (approximately 3.5 μg of protein per lane). The positions of molecular weight markers are indicated on the left. B, gel filtration analysis of purified MJ run as described under “Experimental Procedures.” Red trace (No ATP), MJ equilibrated with 1 mm EDTA and no ATP and eluted without ATP; blue trace (ATP/ATP column), MJ equilibrated with 1 mm ATP and 1 mm EDTA, injected into the column pre-equilibrated with 1 mm ATP, and eluted with the same solution; black trace (ATP/ATP-free column), MJ equilibrated with 1 mm EDTA and 1 mm ATP and eluted without ATP. A₂₈₀ is the absorbance measured at 280 nm normalized to the total absorbance area. C, gel filtration analysis of purified MJI. MJI was equilibrated with 1 mm EDTA and zero ATP (red), 5 μm ATP (black), 10 μm ATP (green), 20 μm ATP (pink), or 40 μm ATP (blue). In all cases the column was pre-equilibrated with ATP-free solution without EDTA, a solution also used for elution. See “Experimental Procedures” for details.

FIGURE 2.

Crystal structure of the MJI ADP sandwich dimer. A, overall dimer structure. Ribbon representation of the two MJI monomers (in green and yellow), with the ADP (white) and P_i (gray) in a space-filling representation and a stick representation of Gln-171 (orange) and Trp-174 (magenta). B, view of the Trp-174 area showing the π-stacked Trps from the NBD monomers.

FIGURE 3.

ATP-dependent changes in Trp-174 fluorescence. A, fluorescence spectra from MJI in the absence (blue) or presence of 2 mm ATP (red). The solutions were nominally divalent cation-free and contained 1 mm EDTA. Excitation wavelength was 295 nm. Buffer-only data were subtracted, and the data were normalized to the peak fluorescence in the absence of ATP. B, dependence of MJI Trp fluorescence on [ATP]. Fluorescence spectra were collected as in A, but varying [ATP] from zero (blue) to 1 mm (red). Intermediate [ATP] were 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 10 (green), 15, 20, 50, 100, 200, and 500 μm. There was a 5-min interval between the sequential ATP additions. Traces correspond to differences with the 2 mm-ATP data and were normalized to the peak emission in the absence of ATP. C, summary of the ATP dependence of MJ and MJI Trp quenching. The fractional maximum fluorescence decrease from experiments similar to that in B is presented as mean ± S.E. The calculated K_d and Hill coefficient were 83.9 ± 8.1 μm and 2.0 ± 0.2 for MJ (n = 4, 3 independent purifications) and 4.2 ± 0.6 μm and 1.7 ± 0.1 for MJI (n = 3, 3 independent purifications).

FIGURE 4.

Time course of dimerization. A, effects of ATP on Trp-174 fluorescence quenching (red, MJ; green, MJI). The signals were normalized to the maximal change. The protein concentration was 2 μm. See “Experimental Procedures” for details. B, time course of Trp-174 fluorescence quenching by ATP after rapid mixing. MJ (red) or MJI (green) were mixed on a stop-flow apparatus with an equal volume of buffer containing ATP, and the decrease in Trp fluorescence was monitored. The final concentrations of protein and ATP were 2 μm and 2 mm, respectively. Black lines are exponential fits to the data (see text). The records in both panels are representative from at least three similar experiments. All solutions were nominally divalent cation-free and contained 1 mm EDTA.

FIGURE 5.

Time course of the monomer/dimer equilibration following ATP hydrolysis. A, effects of ATP and MgATP on MJ (red) and MJI (black) Trp-174 fluorescence. The first (black) and second (red) Mg-labeled arrows indicate the addition of MgCl₂ to MJI and MJ, respectively. The final concentrations of MJ or MJI, ATP, and Mg were 2 μm, 2 mm, and 10 mm, respectively. B, time course of the Trp-174 fluorescence increase elicited by MgATP after rapid mixing. MJ incubated with 2 mm ATP was mixed on a stop-flow apparatus with an equal volume of buffer containing MgATP, and the changes in Trp fluorescence were followed. The final concentrations of MJ, ATP, and Mg were 2 μm, 2 mm, and 10 mm, respectively. The black line is a single-exponential fit to the data. C, response of MJ Trp-174 fluorescence to low [MgATP]. MJ was exposed to 10 mm Mg and then 5 μm ATP followed by 2 mm ATP. The concentrations of MJ and MJI in all panels were 2 μm, and the records are representative of data from at least four similar experiments.

FIGURE 6.

Effect of Mg on the monomer/dimer equilibration. A, time course of MJ Trp-174 fluorescence quenching by ATP (green) and MgATP (red) after rapid mixing. Unlabeled MJ was mixed on a stop-flow apparatus with an equal volume of buffer with ATP alone or MgATP, and the decrease in Trp fluorescence was followed. The final concentrations of MJ, ATP, and Mg were 2 μm, 2 mm, and 10 mm, respectively. Data were normalized to the total Trp quenching, which for MJ in MgATP was ∼50% of the other values (see Fig. 5A). B, time course of MJI Trp-174 fluorescence quenching by ATP (cyan) and MgATP (pink) after rapid mixing. The experimental protocol was the same as that in A. The concentration of MJI was 2 μm. The black lines in A and B are exponential fits to the data (see text). The records in A and B are representative data from at least three similar experiments.