Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

Figures

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  Figure 1. The actomyosin system. (A) Scheme of the actomyosin motor cycle. The myosin motor is subject to conformational changes of the subdomains upon interaction with both the actin filament and the nucleotide adenosine triphosphate (ATP), as well as during nucleotide hydrolysis, and release of its hydrolysis products inorganic phosphate (Pi) and adenosine diphosphate (ADP) to eventually produce force in a two-step mechanism. Color code: subdomains N-terminal domain (pink), U50 kDa (blue), L50 kDa (green), converter (orange), lever arm (grey). (B) Structure of the nucleotide-free Dd myosin-II motor domain, possessing a closed actin-binding cleft, a twisted transducer (cyan), and the converter (orange)/lever arm (grey) in the down position. The active site with the nucleotide sensors P-loop (yellow), switch-1 (red), and switch-2 (brown) is empty and prepared for ATP binding.

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  Figure 2. X-ray crystal structure of the Dd myosin-II motor domain in complex with ADP and blebbistatin. (A) Overview of the crystal structure in the blebbistatin-bound ADP-release conformation. The P-loop together with the bound ADP (grey) is shifted towards the surface. Blebbistatin (magenta) is buried in the known allosteric binding pocket at the apex of the large actin-binding cleft. (B) The 2Fo-Fc density map of ADP in its new binding position. The map was contoured at 1.0 σ. Note that the β-phosphate has rotated away from the P-loop. (C) 2Fo-Fc density map of blebbistatin at the apex of the actin-binding cleft and near the relay helix. The map was contoured at 1.0 σ.

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  Figure 3. The blebbistatin-bound myosin-II·ADP structure (blue) feature characteristics of a myosin conformation between the pre-power stroke (black), and the rigor-like states (grey). (A) The transducer is partially twisted, particularly β-strands 1 to 3. (B) The relay helix and the converter approached the rigor-like position. The converter/lever arm must undergo a ~5° rotation to transition to the final rigor down position (grey). The N-terminal domain is markedly shifted towards its rigor position. (C) The large actin-binding cleft is found in a position between pre-power stroke (black) and rigor-like states (grey), thereby creating a new actin-binding interface. (D) The active site P-loop has moved ~9 Å from its pre-power stroke position in the blebbistatin-bound myosin-II·ADP conformation, but has not reached the rigor position.

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  Figure 4. The myosin-II·ADP·blebbistatin crystal structure shows unique conformational features in the active site. (A) ADP interacts with its phosphate groups, primarily with the reoriented P-loop. The β-phosphate is partially unbound and oriented towards the surrounding water at the protein surface. The adenosine base of the nucleotide is nestled into a groove, formed by a salt-bridge between Arg131 and Glu187. (B) Close-up view on the active site in the myosin-II·ADP·blebbistatin structure shows the critical salt-bridge between switch-1 (Arg238) and switch-2 (Glu459), as well as the newly identified salt-bridge between Arg131 and Glu187 that stabilizes ADP in the release position. A third, complex salt-bridge between Arg232 (switch-1), Asp674 (SH2-helix), and Glu180 (P-loop) seems to be important for mediating the chemomechanical coupling between the active site, the actin-binding region and the mechanical converter/lever arm. (C) Close-up view on the active site in the rigor-like, nucleotide-free myosin-II structure. Due to slight adjustments to the active site, the interaction network around Arg232 is altered as compared to the myosin-II·ADP·blebbistatin structure, and Arg232…

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  Figure 5. Binding assays of ADP to myosin-II using microscale thermophoresis. (A) The affinity of ADP for myosin-II in the absence of magnesium is reduced from KD = 37.5 ± 7.3 µM (10 mM Mg2+) to 145.8 ± 19.3 µM (0 mM Mg2+). (B) Addition of 100 µM blebbistatin increased the binding affinity of ADP for myosin-II, both in the presence (KD = 21.2 ± 3.4 µM) and absence of magnesium (KD = 40.6 ± 3.2 µM).

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  Figure 6. Classical molecular dynamics (cMD) simulations support the stabilizing effect of blebbistatin on the crystallographically determined conformation of myosin-II. (A) Root mean square deviations of the protein backbone atoms, averaged over duplicate 500 ns cMD simulations of blebbistatin-bound (magenta) and -unbound (blue) myosin, confirm the stabilizing effect of blebbistatin on the crystallized conformation, while in the absence of blebbistatin, larger deviations from the start structure were observed. (B) Blebbistatin remains embedded in its binding pocket throughout the simulations, showing only negligible deviations from its crystallized binding pose. (C) Root mean square deviations of the converter indicate no movement of the converter while blebbistatin is bound (magenta); however, a smaller shift towards the pre-power stroke position of the converter is monitored in the absence of blebbistatin (blue). Shown in diagrams A-C are the averages of duplicate cMD simulations. (D) The positions of active site phosphate sensors are stabilized spatiotemporally along the cMD simulations in the presence of blebbistatin, with the critical salt-bridge formed. Binding of ADP in the…

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  Figure 7. Pulling MD simulations using biasing forces on the converter domain visualize coupled conformational transitions in myosin and ADP dissociation. (A) Targeted molecular dynamics (TMD) simulations of the converter rotation from the up position of a strong-ADP-bound myosin conformation with closed actin-binding cleft and partially twisted transducer (light blue) towards the down position (dark blue) were coupled to rearrangements of the N-terminal domain. Structural changes are indicated by vectors. (B) The induced converter rotation during the TMD simulations led to a significant ~6 Å shift of the P-loop towards the front entrance of the active site. The bound ADP moved together with the P-loop towards the surface and lost interactions, which finally led to larger fluctuation of the nucleotide, and the population of a conformation similar to the myosin-II·ADP·blebbistatin crystal structure. (C) Visualization of the release vectors used to pull the nucleotide out of the crystallized position in the myosin motor domain. (D) Pulling forces required to dissociate ADP from myosin along the four different vectors. Independent of the release vector, a consistent set of interactions was…

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  Figure 8. Unbiased MD simulations captured a potential ADP release pathway. (A) Molecular events during ADP dissociation along the unbiased MD trajectories reveal a guiding mechanism of Arg131 together with Thr186. Panels I through III show snapshots along the cMD trajectory. (B) Distance of the protein residues Arg131 and Thr186 to the nucleotide β-phosphate over the MD simulations illustrate the interplay between the two protein residues in the proposed ADP release mechanism. (C) Energy profile of the reconstructed ADP release pathway from a strongly bound ADP to nucleotide dissociation as determined by the molecular mechanics/generalized-born surface area (MM/GBSA) interaction energy between the protein and ADP, obtained by combining TMD and cMD trajectories. Both the myosin conformation with strongly bound ADP (orange dot) and the myosin conformation resembling our myosin-II·ADP·blebbistatin structure (blue dot) represent energy minima (low-energy conformations) along the reconstructed myosin power stroke.