ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation

Abstract

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a C-terminal regulatory domain. The two nucleotide-binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg(2+). ATP was hydrolyzed in the crystallization drop, and ADP-Mg(2+) was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport.

Fig. 1.

Dimerization assays of MalK by gel filtration. (A) Concentration-dependent dimerization in the absence of nucleotides. MalK at a 20 μM concentration (peak fraction) elutes at the expected position of a dimer, whereas at a concentration of 0.1 μM MalK elutes as a monomer. (B) Nucleotide-dependent dimerization at monomeric protein concentration. MalK stock at 1 μM concentration was mixed with 0.5 mM ATP (solid line) or 0.5 mM ADP and 10 mM MgCl₂ (dashed line) and loaded to a size-exclusion column preequilibrated with buffer containing the corresponding nucleotide. The elution peak fraction contains MalK at ≈0.1 μM concentration. Note that in both A and B, for the purpose of comparison, the profiles do not use the same scale. Absorbance is shown in mAU. Figures were prepared with sigmaplot(Systat, Point Richmond, CA).

Fig. 2.

Ribbon diagram of the structure of E. coli. MalK homodimer with bound ADP-Mg²⁺.(A) Schematic diagram showing the subunit structure of a monomer. The NBD (residues 1–235) is in green/cyan and RD (residues 236–371) in yellow. Different colors further distinguish the subdomains or segments in the NBD as follows: green, RecA-like subdomain (residues 1–87 and 152–235); cyan, helical subdomain (residues 88–151). (B) Stereoview of the homodimer viewed down the local twofold axis. ADP is represented in ball-and-stick model (O atoms, red; N atoms, blue; C atoms, yellow; P atoms, orange; and magnesium, purple). Walker A motif is colored in red. The color schemes for the domains of the two monomers are similar, except that one is rendered in lighter color. (C) Stereoview of the homodimer obtained by a 90° clockwise rotation of the structure shown in B about a horizontal axis. Missing residues are shown as dashed lines. Figures were prepared with pymol (www.pymol.org)

Fig. 3.

ADP binding. (A) Stereoview of the electron density (2σ contour level) of one of the bound ADP-Mg²⁺ obtained from a simulated annealing F_o – F_c map, with ADP-Mg²⁺ molecules omitted in the structure factor calculation. The ADP-Mg²⁺ is represented in ball-and-stick model. Water molecules are colored in pink. To illustrate that there is no density in the expected position of the γ phosphate, the ATP molecule also is shown in gray. The ATP model was obtained by aligning the Walker A motif of the ATP-bound form (12) with that of the ADP-bound form. (B) Atomic details of the interaction between MalK and ADP-Mg²⁺. Residues contacting the ADP and magnesium ion are labeled. Hydrogen-bonding and salt-bridge interactions are marked by dashed lines in orange and blue, respectively. Color identification for the residues is as follows: O atoms, red; N atoms, blue; C atoms, gray; and P and S atoms, orange.

Fig. 4.

Conformational changes of MalK upon ATP hydrolysis. (A and B) The nucleotide-binding site of the ATP-bound dimer structure, with ATP sandwiched between the Walker A and B motifs of one monomer and the LSGGQ motif of the other monomer (A) and the posthydrolysis ADP-bound structure (B). The color scheme is similar to that of Fig. 2. (C) Stereoview of superimposed MalK structures in the ATP-bound (red) and ADP-bound (blue) forms. The RDs are rendered in lighter color than the NBDs. ATP (pink) and ADP (cyan) molecules are shown in ball-and-stick model.

Fig. 5.

Three conformations of the MalK dimer. In the resting state, the two NBDs of MalK are separated from each other. In the ATP-bound state, the NBDs have closed, permitting ATP hydrolysis to occur. ATP hydrolysis and release of P_i open the dimer at the NBD interface. Finally, ADP is released from MalK.