Modulation of post-powerstroke dynamics in myosin II by 2'-deoxy-ADP

Abstract

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle.

Keywords: Allostery; Molecular dynamics simulation; Myosin.

Figure 1:. Structural Schematic & Sequence of Myosin.

(A)The structure of the myosin head is highly conserved and can be divided into four subdomains: the N-terminal (tan), upper 50 kDa (teal), lower 50 kDa (brown), and converter (light blue) domains, and these four subdomains interface at the transducer (purple). (B) This schematic illustrates the major steps in the cross-bridge cycle. (1) The cycle begins when (d) ATP binds to myosin heads, releasing them from actin. (2) Next, (d)ATP hydrolysis primes myosin to bind actin. (3) Then, the power stroke occurs. (4) Finally, (d)ADP is released from the binding pocket. (C) The amino acid sequence is represented as a sequence of secondary structure elements (horizontal grey bars: loops, short boxes: β-strands, tall boxes: α-helices). The secondary structure elements are colored according to their respective subdomains in panel A.

Figure 2:. Conformational sampling of ADP and dADP.

Six dihedral angles were defined for both nucleotides: θ₁ (O1B-PB-O3A-PA), θ₂ (PB-O3A-PA-O5’), θ₃ (O3A-PA-O5’-C5’), θ₄ (PA-O5’-C5’-C4’), θ₅ (O5’-C5’-C4’-O4’), θ₆ (O4’-C1’-N9-C8) (A). These angles were organized into three maps for ADP (B) and dADP (C). Each map is a two- dimensional histogram (5°×5° bins) of the conformations sampled by the nucleotides in the nucleotide + myosin simulations (data from the three replicate simulations are combined). The color of each bin indicates the percent of the ensemble that sampled the bin.

Figure 3.. Nucleotide-Myosin interactions.

(A) Each bar corresponds to an interaction between ADP (black bars) or dADP (blue bars) and residues in myosin (x-axis). The height of each bar is proportional to the percentage of the simulation time (averaged across the three replicate simulations) that the contact was observed, error lines denote standard deviation. In panels B-D, residues in myosin are colored according to the difference in contact frequency between ADP and dADP. Residues that interacted more frequently with ADP are colored black and residues that interacted more frequently with dADP are colored blue. The average differences in contact frequency were mapped onto the minimized starting structure (B) and the endpoints of one myosin+ADP simulation (C) and one myosin+dADP simulation (D).

Figure 4:. Conformational sampling in MD simulations of post-powerstroke myosin.

The C_α RMSD of the myosin heads relative to the minimized starting structure was calculated as a function of time (A: ADP; B: dADP) and residue number (C,E: ADP; D,F: dADP). In A-D, results from replicate simulations are shown in shades of grey (ADP) or blue (dADP). In C-D, the average results from the replicate simulations are plotted as a continuous, thick back line. The average C_α RMSF values per residue are mapped onto the minimized crystal structure in panels E and F. (G) After alignment of the trajectories to the most stable residues, we also calculated the average RMSD (averaged both across the duration of the simulation and across replicate simulations) for specific subdomains in myosin. The five regions were the N-terminal domain (10–15, 99–112, 137–142, and 155–168), SH3 domain (residues 34–37, 48–55, 59–63, 70–73, and 77–80), upper 50 kDa domain (residues 210–226, 265–268, 278–287, 290–295, 301–303, 320–334, 338–356, 373–382, 386–394, 411–440, 594–601, 606–613, 615–618) the lower 50 kDa domain (residues 466–494, 506–534, 540–551, 567–580, 584–589, and 630–647), and the converter domain (residues 722–730 and 748:753) Those average values are reported for the ADP (black bars) and dADP (blue bars) simulations.

Figure 5:. dADP-induced conformational changes in the actin binding cleft.

The panels include ribbon structures of the myosin head for the minimized X-ray structure (left) and representative snapshots from MD simulations with ADP (center) or dADP (right). For clarity, the structures of myosin have been clipped such that the portion of the structure between the nucleotide binding pocket and converter domain is not visible here. The myosin heads are oriented with the actin binding cleft facing forward and the nucleotide binding pocket on the right. The nucleotides are represented as spheres. Residues in the lower 50 kDa domain that were associated with the relative twisting motion of the upper and lower 50 kDa domains are colored magenta.

Figure 6:. The structural features of the actin binding cleft were nucleotide-dependent.

Histograms of the solvent accessible surface area of polar atoms within charged residues at the actin binding surface (A), nonpolar atoms within actin binding residues (B) and of specific residues within loop 2 (C) show that the actin binding surface of myosin changes when myosin is bound ADP (black bars) versus dADP (blue bars). Similarly, the geometry of the actin binding cleft (D), as monitored by interatomic distances at 6 residues, was nucleotide dependent. The colors in D are the same as the other panels and the thick vertical bars correspond to the average ADP and dADP distances.

Figure 7:. dADP influences the residue-residue interaction network in myosin.

(A)Intra-residue contacts in myosin that had significantly different frequencies in the ADP and dADP simulations are mapped onto the minimized crystal structure. Those contacts are represented as pipes and are colored black when observed more frequently in the ADP simulations or blue when observed more frequently in the dADP simulations. The pipe diameter is proportional to the difference of the contacts’ frequency in the ADP or dADP simulations. All contact differences involving ADP or dADP are also mapped, independent of statistical significance. (B) Snapshots the minimized X-ray structure (left), and the endpoints of an ADP (middle) and dADP (right) simulation highlight the influence of the bound nucleotide on contacts made by R238, S456, and E459.