High-Speed Atomic Force Microscopy Reveals Rotary Catalysis of Rotorless F ₁ -ATPase

Abstract

One-sentence summary:Intrinsic cooperativity engenders cyclical propagation of conformational states in the stator ring of an ATP-driven rotary motor. 2 ABSTRACTF 1 is an ATP-driven motor in which three torque-generating β subunits in the α 3 β 3 stator ring sequentially undergo conformational changes upon ATP hydrolysis to rotate the central shaft γ unidirectionally. Although extensive experimental and theoretical work has been done, the structural basis of cooperative torque generation to realize the unidirectional rotation remains elusive. We use high-speed atomic force microscopy to show that the rotor-less F 1 still "rotates"; in the isolated α 3 β 3 stator ring, the three β subunits cyclically propagate conformational states in the counterclockwise direction, similar to the rotary shaft rotation in F 1 . The structural basis of unidirectionality is programmed in the stator ring. These findings have implications for cooperative interplay between subunits in other hexameric ATPases.3 F 1 -ATPase, a water-soluble portion of ATP synthase (1), is a rotary motor protein. The α 3 β 3 γ subcomplex (referred to here as F 1 ) suffices as the motor, in which the rotor γ subunit rotates in the stator α 3 β 3 ring upon ATP hydrolysis (2). The concept of the "rotary catalysis" of F 1 was proposed based on biochemical studies (3). It was strongly supported by the first crystal structure (4) and directly proven by observations of rotating single molecules (5). In F 1 , the catalytic sites are located at the α-β interfaces, mainly on the β subunits. In the crystal structure (4), three catalytic sites are in different nucleotide-bound states; one binds to an ATP analog (α TP -β TP in Fig. 1E), another binds to ADP (α DP -β DP ), and the third is unbound (α E -β E ). Both β TP and β DP assume the closed conformation, swinging the C-terminal domain toward γ, whereas β E assumes the open conformation, swinging the domain away from γ. As these two general conformational states appear to push or be pushed by γ, respectively, it was proposed that interactions with γ control the conformational and catalytic states of individual βs to sequentially generate torque (6). In fact, some biochemical studies are thought to suggest that the α 3 β 3 ring alone does not possess intrinsic cooperativity and γ mediates the interplay among βs (7-9). This view was reinforced by studies showing that backward mechanical rotation of γ with external force reverses the chemical reaction toward ATP synthesis (10, 11), whereas forced forward rotation results in accelerated ATP binding (12). 4Recently, however, this contention has been challenged by the finding that even when most interaction sites between β and γ are abolished, F 1 retains catalytic power to rotate γ unidirectionally (13, 14). A few biochemical studies also suggest the intrinsic cooperativity of the α 3 β 3 ring (15, 16). However, as conventional single-molecule optical microscopy requires attachment of a probe onto the rotary shaft for visualization (5), it does not allow direct examin...

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

“…S6). Furthermore, under the imaging conditions used, we estimate the applied force to be too low to affect molecular movement ( 50 pN; refs 21,22), and the tip interacts with the sample only 1.6 times on each pixel over 160 ns. Additionally, we ruled out scan direction bias by increasing the HS-AFM imaging speed to 204 ms per frame, and found similar molecule dynamics ( Supplementary Fig.…”

What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

“…4.5 The concerted kinetics in the ring subunits drives a unidirectional rotation kinetics even in the absence of a rotor shaft High-speed AFM measurements (Uchihashi et al 2011) in a rotorless F 1 -ATPase demonstrated that the opening-closing kinetics of the β subunits follow a unidirectional counterclockwise rotation pattern. The result is consistent with the kinetic scheme in the counter-clockwise rotation observed in the complete F 1 , albeit the individual steps in the rotorless F 1 -ATPase are slower.…”

“…However, when considering higher nucleotide concentrations, in particular in rotation experiments for F 1 -ATPase under physiologically relevant conditions, giving rise to a multi-site activity, events of binding/release and hydrolysis/ synthesis occurring in pockets of different β subunits are directly coupled. These inter-subunit interactions are strong enough to determine the directional rotation kinetics even without the γ shaft (Uchihashi et al 2011), or a truncated shaft (Furuike et al 2008). So while the angular position θ of the latter does affect the rates of the processes in the β subunits, it is not essential in mediating the interactions between the β subunits that lead to the concerted kinetics.…”

“…3. As described earlier, high-speed AFM measurements were used to monitor the rotary kinetics of opening and closing of the β subunits (Uchihashi et al 2011). As in item 2 above, in these experiments the empty subunits awaiting the binding of an ATP were in an open state, while the other two subunits, presumably with nucleotides in their catalytic pockets, were in a closed state.…”

Operation mechanism of FoF1‐adenosine triphosphate synthase revealed by its structure and dynamics

“…The importance of the c-subunit in the rotary catalysis of F 1 has been directly examined using high-speed atomic force microscopy (AFM) (31), by observing the conformational changes of the three b-subunits in the stator a 3 b 3 ring without the c-subunit (32,33). In contrast to the nucleotide-free (Fig.…”