Mechanism of substrate translocation by a ring-shaped ATPase motor at millisecond resolution

Abstract

Ring-shaped, hexameric ATPase motors fulfill key functions in cellular processes, such as genome replication, transcription, or protein degradation, by translocating a long substrate through their central pore powered by ATP hydrolysis. Despite intense research efforts, the atomic-level mechanism transmitting chemical energy from hydrolysis into mechanical force that translocates the substrate is still unclear. Here we employ all-atom molecular dynamics simulations combined with advanced path sampling techniques and milestoning analysis to characterize how mRNA substrate is translocated by an exemplary homohexameric motor, the transcription termination factor Rho. We find that the release of hydrolysis product (ADP + Pi) triggers the force-generating process of Rho through a 0.1 millisecond-long conformational transition, the time scale seen also in experiment. The calculated free energy profiles and kinetics show that Rho unidirectionally translocates the single-stranded RNA substrate via a population shift of the conformational states of Rho; upon hydrolysis product release, the most favorable conformation shifts from the pretranslocation state to the post-translocation state. Via two previously unidentified intermediate states, the RNA chain is seen to be pulled by six K326 side chains, whose motions are induced by highly coordinated relative translation and rotation of Rho's six subunits. The present study not only reveals in new detail the mechanism employed by ring-shaped ATPase motors, for example the use of loosely bound and tightly bound hydrolysis reactant and product states to coordinate motor action, but also provides an effective approach to identify allosteric sites of multimeric enzymes in general.

Figure 1

Structure and proposed rotary reaction mechanism of Rho hexameric helicase. (a) Structure modeled based on the reported Rho crystal structure (pdb code: 3ICE). Rho subunits consist of a C-terminal domain (blue) and an N-terminal domain (grey). Active sites of the ATPase cycle are located at the subunit-subunit interfaces and are labeled according to the state of ATP at the respective sites, namely, hydrolysis-competent (T*), ATP-bound (T), “old” product (D) and empty (E) state, respectively. ATP, ADP and Pi are shown in orange. K326 residues (purple), contributed by each subunit and altogether arranged in a roughly helical stair case fashion, are located in the central channel of the Rho hexamer where RNA (pale red) is bound, likewise in a roughly helical conformation. (b) Proposed rotary reaction mechanism. Rho’s six identical subunits are labeled m₁, m₂, …, m₆; the different colors of the six subunits’ circular peripheries indicate schematically that the subunits assume different interface conformations in prior state R. In binding ATP, releasing ADP+Pi and hydrolyzing ATP to ADP, the pattern of ligand binding states corresponding to the surface conformations in the prior state R, T*T*TTED, shifts to DT*T*TTE in the final state F. As one can easily recognize, the ligand binding states of R, after a 60° clockwise rotation around the z-axis, are the same as those of F. In going from R to F, RNA is propelled as indicated by pale (RNA further away from viewer) and intense red (RNA closer to viewer) coloration.

Figure 2

Free energy profiles and mean first passage times characterizing the rotatory reaction step of Rho. (a) Rotary reaction scheme. Details of how states R, I and F have been constructed are described in Methods. The ligand state X at the interface between subunits m₆ and m₁ is assumed to be in one case the empty state E and in another case the (“old”) product bound state D. The free energy profiles during the transition I → F for cases X = E and X = D are shown in (b) and (c), respectively. The standard error shown is estimated by repeating the free energy calculations with half of the trajectories. Insets in (b) and (c) show both the forward and backward mean first passage times. In case X = E the rotary reaction is exergonic whereas in case X = D the rotary reaction is strongly endergonic.

Figure 3

Relative translational and rotational motion of subunits during the I → F transition. (a) Projection of subunit positions (center of mass of each subunit’s atoms in the collective variable set) on the z-axis as a function of the progress along the discrete reaction coordinate (0, 1, …, 50), namely the image numbering representing the progress of the transition pathway. (b) Front view of the six subunits in the initial state I (left) and in the final state F (right). The motion of m₆ along the z-axis is indicated by a blue arrow. The subunit colors are consistent with (a). (c) Two-dimensional free energy landscape generated using the contact area between m₁ and m₆ (σ₆₁) and the crossing angle between the principal axes of m₁ and m₆ (θ₆₁) as coordinates. The unit for the free energy is kcal/mol. States I, IM1, IM2 and F (as labeled in Figure 2b) are shown as black dots. Insets demonstrate the relative orientations between m₁ and m₆ in state I (bottom left inset) and in state F (top right inset).

Figure 4

Key interactions between RNA and Rho during the I → F transition. (a) Free energy landscape along distances P₃-K326(m₁) and P₆-K326(m₄). The position of the phosphorus atoms of the phosphate group and NZ atoms of K326 define the corresponding distances. (b–e) Configurations in states I, IM1, IM2 and F, accounting for the key steps of RNA binding and release that induce RNA translocation. Red dashed arrows indicate large side chain movements before the next state is reached. m₁ (green) and m₄ (orange) are shown in transparent surfaces.

Figure 5

Free energy landscape for RNA translocation distance and Rho conformation for X = E. The energy values are defined by the color bar in units of kcal/mol. States I, IM1, IM2 and F are labeled with black dots. The RNA translocation distance is defined as the center of mass distance between RNA backbone (residues 3 – 8) and Rho backbone (C-terminal domain). The Rho conformational coordinate is defined in the text.

Figure 6

Allosteric coupling between RNA translocation and Pi of the hydrolysis product. The coupling is established through a link between the disengagement of K326(m₁) from RNA phosphate P₃ and release of Pi from a salt bridge with R269(m₁). The figure shows in (a, b, c, d corresponding to states I, IM1, IM2, F, respectively) top-down views (same view of Rho as in Figure 1a) the conformational changes of residues R272(m₁), E333(m₂), E334(m₂), and R269(m₁) at the m₁/m₂ interface during transitions I → IM1 → IM2 → F. The three transitions are shown as black arrows; red arrows indicate large side chain movements occurring before the next transition takes place. RNA is shown as a pink ribbon with phosphate P₃ highlighted as a tan sphere.