Two ATPases

Abstract

In this article, I reflect on research on two ATPases. The first is F(1)F(0)-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization.

FIGURE 1.

Trp probes in the F₁ catalytic site. Residues located around MgAMPPNP (an analog of MgATP; shown in green) in the catalytic site of F₁ were substituted with Trp. Residues indicated in yellow gave the same fluorescence response to MgADP and MgATP, valuable for measuring binding affinity and occupancy. Residues indicated in red responded differently to MgADP versus MgATP. β-Trp-297 and α-Trp-291 responded specifically to site one (highest affinity), yielding proof that this site persists in steady-state hydrolysis. The residue shown in gray did not respond to nucleotide. This figure was reprinted from Ref. with permission.

FIGURE 2.

Mechanism of ATP hydrolysis by F₁. From Ref. . In 2004, we combined our original mechanism (26, 35) with rotational information from single-molecule experiments of M. Yoshida and K. Kinosita. Catalytic sites (circles) are designated O for open and H, M, or L for high, medium, or low affinity for MgATP. In a series of enzyme states (ABCDA), one MgATP is consumed, and the γ-subunit rotates 120°. Panel a, binding of red ATP generates rotation of γ, hydrolysis of already-bound green ATP, and a switch in site conformations (“binding change”). Already-bound blue ADP is then released. Note that red ATP binding, green ATP hydrolysis, and blue ADP release occur at three different catalytic sites. Panel b, a new (black) ATP binds and brings about the same set of events, with this time prebound red ATP being hydrolyzed and green ADP being released. Panel c, finally, in a third series, the red ADP product is released.

FIGURE 3.

Mg²⁺ coordination in the catalytic site of F₁. As deduced in Ref. . Green sphere, Mg²⁺; cyan spheres, water molecules in the first coordination shell. Residues involved in Mg²⁺ coordination are β-Thr-156, β-Glu-185, and β-Asp-242. The catalytic carboxylate β-Glu-181 is not involved, although it is in close proximity. This figure was reprinted from Ref. with permission.

FIGURE 4.

The F₁ catalytic transition state. As deduced in Ref. . Ligands to the pentacovalent phosphorus are shown, with the reactant water molecule in cyan, the β-subunit in gray, and the α-subunit in green. α-Arg-376 is the arginine finger. This figure was reprinted from Ref. with permission.

FIGURE 5.

E. coli F₁F₀. This structure (based on Ref. 87) is a composite from x-ray structures and molecular modeling. The stator stalk consists of subunits b₂ and δ, interacting with subunits a and α, respectively.

FIGURE 6.

Binding of the δ-subunit to the N terminus of the α-subunit. Shown is the binding of a 22-residue peptide corresponding to the N-terminal region of the α-subunit to the δ-subunit as deduced by NMR (65). The helical peptide (gray) nestles between two helices (blue and orange) on δ. The naturally occurring δ-Trp-28 provided an excellent optical probe.

FIGURE 7.

P-glycoprotein alternating sites mechanism. Rectangles, transmembrane domains; circles, squares, and hexagon, different conformations of the N- and C-terminal catalytic sites (NBDs); black circles, drug molecule. Binding of ATP to an empty N-terminal site (upper left) brings about hydrolysis of ATP in the C-terminal site (upper right). Relaxation of the C-terminal site drives drug from inward-facing higher affinity to outside-facing lower affinity (lower right) as P_i is released. Drug and ADP dissociate (lower left), and in the next cycle, it will be the C-terminal site that binds ATP and the N-terminal site that hydrolyzes it. This figure was reprinted from Ref. with permission.

FIGURE 8.

The occluded nucleotide conformation in the P-glycoprotein catalytic pathway. From Ref. . Each half-circle represents an NBD. The occluded nucleotide conformation occurs as a closed NBD dimer at step III, with the ATP that is tightly bound and committed to hydrolysis shown in boldface. Steps inhibited by mutations and covalent reagents or accelerated by drug are shown. NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; NEM, N-ethylmaleimide.