Steps of actin filament branch formation by Arp2/3 complex investigated with coarse-grained molecular dynamics

The nucleation of actin filament branches by the Arp2/3 complex involves activation through nucleation promotion factors (NPFs), recruitment of actin monomers, and binding of the complex to the side of actin filaments. Because of the large system size and processes that involve flexible regions and diffuse components, simulations of branch formation using all-atom molecular dynamics are challenging. We applied a coarse-grained model that retains amino-acid level information and allows molecular dynamics simulations in implicit solvent, with globular domains represented as rigid bodies and flexible regions allowed to fluctuate. We used recent electron microscopy structures of the inactive Arp2/3 complex bound to NPF domains and to mother actin filament for the activated Arp2/3 complex. We studied interactions of Arp2/3 complex with the activating VCA domain of the NPF Wiskott-Aldrich syndrome protein, actin monomers, and actin filament. We found stable configurations with one or two actin monomers bound along the branch filament direction and with CA domain of VCA associated to the strong and weak binding sites of the Arp2/3 complex, supporting prior structural studies and validating our approach. We reproduced delivery of actin monomers and CA to the Arp2/3 complex under different conditions, providing insight into mechanisms proposed in previous studies. Simulations of active Arp2/3 complex bound to a mother actin filament indicate the contribution of each subunit to the binding. Addition of the C-terminal tail of Arp2/3 complex subunit ArpC2, which is missing in the cryo-EM structure, increased binding affinity, indicating a possible stabilizing role of this tail.

Movie 3 Serial simulation of a single actin subunit bound to the Arp3 of active Arp2/3 complex without VC domain ( Figure S5A). The simulation was performed in a cubic box of side 300Å for 1µs.
Movie 4 Serial simulation of a single actin subunit bound to the Arp2 of active Arp2/3 complex without VC domain ( Figure S5B). The simulation was performed in a cubic box of side 300Å for 1µs.
Movie 5 Serial simulation of a single actin subunit bound to the Arp3 of active Arp2/3 complex in the presence of VC domain ( Figure S6A). The simulation was performed in a cubic box of side 300Å for 1µs.
Movie 6 Serial simulation of a single actin subunit bound to the Arp2 of active Arp2/3 complex in the presence of VC domain ( Figure S6B). The simulation was performed in a cubic box of side 300Å for 1µs.
Movie 7 Serial simulation of the active Arp2/3 complex bound to the mother actin filament, with ArpC2 C-terminal tail built between ArpC1 protrusion loop and ArpC4 ( Figure 5). The simulation was performed in a cubic box of side 400Å for 1µs.
Movie 8 Serial simulation of the active Arp2/3 complex bound to the mother actin filament, with ArpC2 C-terminal tail built between ArpC1 protrusion loop and Arp3 ( Figure S10). The simulation was performed in a cubic box of side 400Å for 1µs. Figure S1. Replica dwell time distribution of the REMD simulation of CA domain and inactive Arp2/3 complex of Figure 1A. Each line shows the distribution of time on different temperatures over the 1.6 µs simulation. The black line is the average of all 28 replicas. The error bars are standard deviations. Replicas span multiple temperatures, indicating the system is approaching equilibrium where the time a replica spends at every temperature site should be similar. Figure S2. Temperature-and time-dependence of the simulation with CA domain and inactive Arp2/3 complex of Figure 1A. 2D distributions of dRMS of C domain to Arp2 binding site and A domain to ArpC1 binding site, as in Figure 1A for the (A) entire simulation, and (B) the last 800 ns (second half of the simulation). At 540 K, the CA domain is essentially always dissociated from the Arp2/3 complex through the entire simulation. The system gets trapped at the lowest energy hotspot in the second half of the simulation at low temperatures (180 K-240 K in panel B). Weaker bound states are observed as transient hotspots at low temperatures in the early part of the simulation (180 K-300 K of panel A) or as weaker hotspots at intermediate temperatures in the second half of the simulation (300 K-420 K of panel B).      Figure 1A and 3C, and of actin with respect to long-pitch binding to Arp2, as in Figure 3A. Left: 2D distribution of dRMS values for actin and C domain relative to Arp2. Middle: 2D distribution of dRMS values for C and A domains relative to their Arp2-ArpC1 binding sites. Right: 2D distribution of dRMS vs interaction energy between actin and Arp2/3 complex. Data binned over 0.5 × 0.5Å 2 or 0.1 kCal/mol × 0.5Å. (C) Same as first plot of panel B, but for VC-actin instead of VCA-actin. VC domain is the same as in Figures 2, 3A and 3B (R431-I481). Both VCA-actin and VC-actin simulations performed in cubic boxes of side 300Å for ∼ 1.8 µs and ∼ 1.4 µs respectively. Figure S8. Delivery of actin to the active Arp2/3 complex without VC domain. REMD simulations of actin and active Arp2/3 complex starting from separate state (snapshot). The 2D-distributions of dRMS and binding energies are calculated and plotted in bins of 0.1 kCal/mol × 0.5Å or 0.5 × 0.5Å 2 . The bound conformations are randomly selected the from the hotspot with the lowest dRMS values. Simulation performed in a cubic box of side 300Å for ∼ 900 ns. Figure S9. Simulation of active Arp2/3 complex bound to a mother actin filament without ArpC2 C-terminal tail. Serial simulation as of Arp2/3 complex interacting with a mother actin filament as in Figure 5C,D, but with the Arp2 C-terminal tail left missing. (A) dRMS of Arp2/3 complex subunits in contact with mother actin filament versus time. (B) Interaction energies of Arp2/3 complex subunits and of whole complex with mother filament versus time. Simulation shows quick movement of Arp2/3 complex away from its initial position as compared to the case with the ArpC2 tail included ( Figure 5). Figure S11. REMD simulation of active Arp2/3 complex and mother actin filament starting from separate state. The Arp2/3 complex and mother actin filament were taken from PDB 7AQK and treated as separate rigid bodies except for the D-loops of the mother actin filament. Unlike Figure 5, Arp2 and Arp3 were set fully rigid. The LAMMPS mass variable of Arp2/3 complex was reduced to allow faster diffusion of the complex. To prevent binding of Arp2/3 complex to mother filament ends, or binding of the barbed ends of Arp2/Arp3 with mother actin filament, repulsive beads (magenta spheres) were added to the ends of the filament and in between Arp2 and Arp3. These beads interact with all other atoms in the simulation through a repulsive only LJ potential of ϵ 10.0 kCal/mol, and σ and cutoff both 42.5Å. The Arp2/3 complex interface with Arp2/Arp3 barbed end, which binds the daughter actin filament, was also capped with a repulsive bead interacting with other atoms through a similar repulsive LJ potential of the same ϵ and cutoff of 34Å. The interaction ranges were selected to properly cap the corresponding interface. In simulations without the addition of repulsive beads, such interactions were occurring with large probability. The size of the spheres reflect the range of the repulsive interaction. Right: 2D distribution of dRMS vs binding energy, evaluated with respect to the 7AQK reference. The Arp2/3 complex associated with the actin filament in several orientations indicated by several hotspots. We did not observe a low dRMS peak, presumably because the interaction energy in the 7AQK reference is weaker without the ArpC2 tail (compare Figure  5D to Figure S7B). Simulation performed in a cubic box of side 400Å for 400 ns. The simulation was repeated four more times with different starting random seeds, giving similar results.