Mechanism of Phosphate Release from Actin Filaments

Figures

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  Figure 1.. Pathways of Pi release in independent WT-MetaD simulations. (Panel A) Cartoon representation of the actin filament 5-mer with the subunits colored in blue, orange, green, red, and purple from A0 at the barbed end to A4 at the pointed end. Our simulations focus on the phosphate release from actin subunit A2, which is highlighted by the black box. (Panel B) One representative trajectory of phosphate release (orange spheres) from actin subunit A2 (green cartoon representation) made by plotting the positions of the phosphorus in each frame. R177 and N111 indicate the exit of the “backdoor” pathway (see Supplementary Fig S1 for details of other Pi release trajectories). (Panel C) Time course of the subunit dihedral angles in WT-MetaD simulations with unrestrained simulations in the left column and restrained simulations in the right column with a force constant of 5000 kJ/mol/radian2 applied on subunits A0, A1, A2 and A4. (Panel D) Potential of Mean Force (PMF) as a function of the distance between phosphate and the Mg2+ ion from the constrained (green) and unconstrained (blue) simulations. The shaded regions are standard errors. The positions of the energy barriers are highlighted by…

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  Figure 2.. The release of Pi proceeds with the solvation of Mg2+ and the disruption of interactions with nearby residues near the active site. (Panel A) A 2-dimensional plot of potential of mean force (PMF) as a function of the number of waters coordinated with Mg2+ and the distance between Pi and Mg2+ calculated from one release trajectory. The bar on the right has the color code for energy levels in kcal/mole. Supplementary Materials Fig. S4 has PMFs from five other trajectories. B-E, Stick figures of the active site with Mg2 depicted as a purple sphere using the VDW presentation with a 0.8 scale, hydrogen bonds as black dashed lines, electrostatic interactions as red dashed lines, and nonbonding distances as blue solid lines. B-C, ADP and Pi at two time points during a WT-MetaD simulation. (Panel B) Mg2+ is coordinated with 3 water molecules. (Panel C) Mg2+ coordinated with 4 water molecules after losing coordination with Pi. D-E, Hydrogen-bonds at two time points during a WT-MetaD simulation. (Panel D) Before dissociating from Mg2+, Pi was coordinated with the oxygen of the side chain of residue S14 and a hydrogen atom of the side chain amine group of residue Q137. (Panel E) After…

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  Figure 3.. Pi diffuses in a cavity inside an actin subunit between the active site and the R177 back door in the occluded state. The cavity is shown as a grey surface defined by probing an equilibrium structure from an unbiased MD simulation in the occluded state with a detection radius of 4.2 Å. Mg2+ is shown as a purple sphere, ADP, G74, A108, N111, R177 are shown in the licorice representation. The S-loop (residues 70 to 79) is highlighted in yellow. The positions of the phosphorus atom of the γ-phosphate at 0.5 ns intervals during in a WT-MetaD simulation are shown as spheres with a gradient of colors ranging from red at early times to yellow at late times. (Panels A-B) Cavity from two viewpoints in the occluded state with orange arrows to indicate the path of the release process. Clusters are present next to Mg2+ and in the cavity leading up to the gate. (Panel C) During the release process, hydrogen bonds (black dash lines) form transiently between Pi (orange stick figure) and the backbone carbonyl oxygen of residue G74 and the amide hydrogen of residue A108.

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  Figure 4.. Hydrogen bonding networks of R177 in the (Panels A-B) closed state, (Panels C-D) occluded state and (Panels D-E) open state. The top row shows stick figures with hydrogen bonds as black dashed lines and longer distances to potential hydrogen bond partners as blue lines. The second row shows surface representations of the residues forming the gate in MD simulations. The surfaces of the sensor loop (residues 71 to 77), R177, N111, and P109 are colored by the charge density with red for positive charges and blue for negative charges. (Panel A) Cryo-EM structure of ADP-Pi-actin filaments (PDB 8A2S) with a hydrogen bonds between R177 and the sidechain of N111 as well as the backbones of HIC73 and P109 closing the gate. (Panel B) Surface representation of the closed state in an MD simulation. (Panels C-D) Snapshots from an unbiased MD simulation in the occluded state. R177 hydrogen bonds with the backbone of P109 and the side chain of H161 precluding the release of phosphate. (Panels E-F) Open state with an electrostatic interaction between the sidechains of R177 and D179 that opens a channel for phosphate to escape. (Panel E) Crystal structure of MAL-RPEL2 complexed to G-actin (PDB…

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  Figure 5.. (Panels A-B) Distances between R177 and its hydrogen bonding partners at 1 ns intervals in four unbiased MD trajectories. The colors of individual data points and the bars at the top of each panel indicate the four states, closed (red), occluded (grey), open (acqua) and wide-open (green). (Panel A) R177-N111. (Panel B) Distances between the sidechain of R177 and the sidechains of N111, H161 and D179 and the backbones of HIC73 and P109. (Panel C) Free energies vs. distances between R177 and residues with which it forms hydrogen-bonds. Orange stars indicate energy minimums and green shaded areas depict regions where hydrogen bonds or salt bridges form between corresponding residue pairs. The energy profiles depend on the distinct chemical properties of the residues and their surrounding environments. For example, the R177-N111 distance has three distinct basins in the energy landscape. The minimum around 2.5 Å, is associated with the hydrogen bond between R177 and N111. The other basins at 8.5 Å and 16.0 Å represent interactions that stabilize the system such as hydrogen bonds with other residues such as P109 and D179.

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  Figure 6.. Unbiased MD simulations employing various temperature schemes. (Panel A) Starting with the closed state the temperature was maintained at 77 K throughout the simulation. (Panel B) Starting with the closed state the temperature gradually decreased from 322 K to 77 K over the initial 40 ns and remained constant at 77 K thereafter. (Panels C-D) The temperature was alternated periodically using a linear decrease from 322 K to 77 K over 2 ns, followed by a constant temperature of 77 K for 1 ns, before a linear increase back to 322 K over 2 ns. (Panel C) Starting with the closed state. (Panel D) Starting with the occluded state. The backdoor gate remained closed state with the temperature schemes in panels A-C and tended from the occluded state toward the closed state in panel D.