Revealing a Hidden Intermediate of Rotatory Catalysis with X-ray Crystallography and Molecular Simulations

Abstract

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remains an unresolved puzzle in biological energy transfer. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multistep rotation process. Here, using vacuolar or V₁-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography, a new intermediate or "dwell" is identified, which enables the release of an inorganic phosphate (or P_i) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the side-chain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. An inspection of all known rates encompassing the 4-step rotation mechanism implicates the overcoming of the ADP interactions with V₁-ATPase to be the rate-limiting step of motor action.

Figure 1

Structure of the 2_(ADP·AlF4)V₁-bound V₁ complex. (A) Side view of 2_(ADP·AlF4)V₁. (B) Top view of the C-terminal domain (shown in panel A at the transparent surface) of 2_(ADP·AlF4)V₁ from the cytoplasmic side. Red arrows indicate the nucleotide-binding sites. The bound ADP and AlF₄^– molecules are shown in a space-filling representation and colored orange and cyan, respectively. Superimposed structure at the N-terminal β-barrel region (white) of three structures of A subunits (C) and B subunits (D) in 2_(ADP·AlF4)V₁. A subunits are colored light blue (A1 or A^e), dark blue (A2 or A^b), and darker blue (A3 or A^t) in order of openness. Similarly for the B subunits: dark purple (B1 or B^e), light purple (B2 or B^b), and darker purple (B3 or B^t). The P-loops are shown in yellow. (E–G) Magnified view of the nucleotide-binding sites of 2_(ADP·AlF4)V₁, corresponding to the red box of panel C. The positions of the nucleotide-binding sites correspond to the symbol written in panel B. The |Fo| – |Fc| maps calculated without ADP:Mg²⁺ and aluminum fluoride molecules at the binding pockets contoured at 4.0 sigma are shown in red (negative) and green (positive).

Figure 2

Comparison of the structures of 2 ADP·AlF₄^–-bound and 2 AMP·PNP-bound V₁complexes. (A, B) The structures of 2_(ADP·AlF4)V₁ (colored) are superposed on the AMP·PNP-“bound” or 2_(AMP·PNP)V₁ conformations (shown in gray). (A) Side view and (B) top view of the C-terminal domain from the cytoplasmic side. The bound AlF₄^– and ADP molecules are shown in space-filling representation and colored cyan and orange, respectively. (C–E) The “empty” (C), “bound” (D), and “ADP·P_i-bound” (E) forms in 2_(ADP·AlF4)V₁ (colored) are superimposed on those of 2_(AMP·PNP)V₁ (shown in transparent gray) at A subunits (residues 67–593). (left) Magnified views of the nucleotide binding sites, corresponding to the green box of middle panels. (middle) Side views of AB pairs. (right) Magnified views of the interface of C-terminal domains, corresponding to the red box of the middle panels. Red (2_(ADP·AlF4)V₁) and black (2_(AMP·PNP)V₁) dotted lines indicate the distances (Å) between C_α atoms. The numbers outside the panel represent the value of the lengths of the red dotted lines minus the lengths of the black dotted lines.

Figure 3

Proposed model of the rotation mechanism of E. hirae V₁-ATPase. (A–C) The structure models are based on the crystal structures of catalytic dwell (2_(AMP·PNP)V₁ in panel A), P_i-release dwell (2_(ADP·AlF4)V₁ in panel B), and ATP-binding dwell (2_ADPV₁ in panel C). The ATP indicated with a yellow terminal P_i is committed to hydrolysis.

Figure 4

ATP hydrolysis breaks the dynamic coupling between A- and B-subunits. (A) ATP is bound to the binding pocket, formed by G235, G237, K238, R262, and F425 of the A-subunit and R350 of the B-subunit, prior to hydrolysis. In this state, the A^t- and B^t-subunits are tightly coupled due to the presence of the ATP. (B) Upon hydrolysis and prior to release of the inorganic phosphate (P_i), the ADP and P_i moieties interact primarily with the A- and B-subunits, breaking the tight coupling seen in panel A. (C) Network model showing correlated movements between the A- and B-subunits before hydrolysis, with R350 of the B-subunit highlighted in blue. Tight binding of the subunits gives rise to a strong network. (D) Correlated movements between the A- and B-subunits after hydrolysis, with R350 of the B-subunit highlighted in blue. Breakage of the tight coupling between the subunits results in a weakly coupled network.

Figure 5

Pathways of P_i release. The two simulated pathways for phosphate release are either (A) outward (away from the stalk) or (B) inward (toward the stalk). The first event of phosphate release captured using funnel metadynamics is shown; P_i molecules are shown as orange spheres with the white arrow depicting the direction of egress. The free energy for the phosphate release computed by funnel metadynamics along the funnel and orthogonal axes is shown for the (C) outward pathway and (D) inward pathway. The outward P_i-release pathway is energetically less expensive. In the vicinity of the binding pocket, the inward pathway is more constrained and encompasses higher barriers following disengagement of P_i from the pocket. Exemplary traces of a diffusive particle on these surfaces determined using BD simulations reveal that traversal of the inward pathway is ∼10-fold slower; BD time steps for probing the inner pathway are 1 ps, and those for the outer are 3 ps, as the former is found to be more rugged (see Figure S7).

Figure 6

Interface energy analysis. Upon P_i release, interaction energy reduces (becomes less negative) between the stalk and subunit A^eB^e (top panel, left), B^eA^t (middle panel, left), and A^tB^t (bottom panel, left). Concomitantly, the solvent-accessible surface area (SASA) of the empty pocket (top panel, right), non-nucleotide binding pocket (middle panel, right), and ADP·P_i-bound pocket (bottom panel, right) undergoes an increase. This increase in SASA of the empty pocket allows it to accept a new ATP molecule to reset the rotatory catalysis cycle.

Figure 7

Coupling scheme for ATP hydrolysis of E. hirae V₁-ATPase. Each cycle in the figure represents the chemical state of the nucleotide-binding site from the cytoplasmic side. The central arrows in the ellipses represent the orientation of the central axis beginning from the 12 o’clock position, which corresponds to the catalytic dwell. ATP* represents an ATP molecule that is committed to hydrolysis. We have identified the final, ADP-release step as rate-limiting. See the text for additional details.