The Complex in Action: Actin Nucleation

The Arp2/3-complex in activated form will promote actin nucleation, causing the growth of a new filament under a 70° angle. The Arp2/3 complex contacts three actin subunits along one helix of the actin mother filament, led by subunits p34 and p40 [1]; as structural data already suggested, the two Arp-subunits are capable of dimerizing with each other and could hence form the first two subunits of the new branch in a similar fashion to actin dimerization, forming a heterodimer with hydrophobic residues, highly conserved between actin and the Arp-subunits, to stabilize the interactions [10]. This process could bypass the kinetically rather unfavourable dimerization of actin: the Arp2/3 complex binds to the side of the actin mother filament and side-branch at the characteristic 70° angle is formed. Several Arp2/3-complex subunits possess sites capable of binding actin directly, which could explain why the Arp2/3-complex seems to promote actin cross-linking as well as branch formation.

The Arp2/3-complex is activated by binding of WASp-protein to the complex, presumably at two different sites; one site on Arp3, and one on the Arp2/APRC1 interface. Additionally, SCAR-proteins are thought to bind the p21-subunit [13]. The actin monomer then attaches to the ‘barbed’ end of Arp2; the three domains of WASp are then orientated in such a way, with the C-terminal tryptophan residue of the A-domain in the pocket between Arp3 and ARPC3, that a second actin subunit can be delivered to the newly growing branch.

Figure 7. From [15] Cooper et al. (2001), who in turn adapted from original by Volkmann et al. (2001). Shown is the Arp2/3-complex involved in actin branch formation, with Arp2 and Arp3 forming the first two subunits of the new branch growing at the new ‘barbed’ end.

Figure 7. From [15] Cooper et al. (2001), who in turn adapted from original by Volkmann et al. (2001). Shown is the Arp2/3-complex involved in actin branch formation, with Arp2 and Arp3 forming the first two subunits of the new branch growing at the new ‘barbed’ end.

The most crucial process in the activation of nucleation activity of the complex is the binding and subsequent hydrolysis of ATP [8], with nucleotides determining the affinity for regulatory proteins, causing structural changes in subunits that allow regulatory proteins to bind or dissociate from the complex. Within the Arp2/3-complex however, the two actin-related proteins, 2 and 3, have minimal contact in the inactive nucleotide-free complex: activation occurs in a multi-stage process, in which the two Arp-subunits are brought together until they resemble two successive actin-subunits. The Arp-dimer then binds an actin monomer to initiate branching, presumably requiring ATP-hydrolysis by Arp2; Arp3 does bind ATP, but hydrolysis was not detected in the inactive or active complex. The first step towards understanding the mechanism through which nucleotide-binding causes activation was identification of the residues crucial for this process, which was done by Martin et al. (2005), see figure 6.

Figure 8. Adapted from Martin et al. (2005) [11] who investigated effects of mutations in the nucleotide-binding pockets of Arp2 and Arp3; indicated are residues that were found to play a crucial role in nucleotide- binding, based on the crystal structure from Robinson et al. [1], with actin-domains substituted for the missing Arp2-subdomains I and II.

Figure 8. Adapted from Martin et al. (2005) [11] who investigated effects of mutations in the nucleotide-binding pockets of Arp2 and Arp3; indicated are residues that were found to play a crucial role in nucleotide- binding, based on the crystal structure from Robinson et al. [1], with actin-domains substituted for the missing Arp2-subdomains I and II.

In order to determine the conformational changes that might be involved in activation of the Arp2/3-complex, Nolen et al. (2004) [12] compared the Arp2/3 complex, and specifically the Arp2 and Arp3 subunits, either nucleotide-free, bound to ATP, or bound to ADP. In both Arps, nucleotides bound to a notch between subdomains 3 and 4 in a similar way to nucleotide-binding to actin, which was to be expected based on the structural/sequence similarities between the Arps and beta-actin, as shown previously.

Nolen et al. assumed presence of calcium ions within the nucleotide-bound structures, based on Omit maps and the fact that conserved actin-residues were found that are known to make water-mediated calcium contacts [8]. Their findings suggest that, although nucleotide binding is necessary, it is not sufficient for actin nucleation to occur.

Figure 9. The Arp2/3-complex with Arp2 in ‘skyblue’ and Arp3 in ‘cyan’. Indicated are the (presumed) calcium ions (orange ‘spheres’) and bound ATP-molecules (red ‘stick’ models).

Figure 9. The Arp2/3-complex with Arp2 in ‘skyblue’ and Arp3 in ‘cyan’. Indicated are the (presumed) calcium ions (orange ‘spheres’) and bound ATP-molecules (red ‘stick’ models).

The Arp3-cleft closes after ATP is bound, with the conformational changes then propagating throughout the complex: this is caused by a 4.1° rotation of subdomains 1 and 2 towards 3 and 4, which resembles a ‘scissor’-motion which also occurs in actin monomers. Subsequently, a second 8.5° rotation causes one a-helix and two b-sheets (12 and 13) from subdomain 4 to tilt towards subdomain 3 to further close the ‘notch’ after nucleotide binding: this rotation is more pronounced with ATP binding compared to ADP binding. Additionally, p16, p40 and part of p20 rotated clockwise, bending the p20-long C helix and bringing p40 closer to Arp2. ADP binding caused relatively little change in the complex conformation; nucleotide-binding to Arp2 did not cause any significant conformational changes. Arp3, although involved in causing the conformational changes possibly necessary for complex activation, shows low ATPase activity: His-176, which is known to be crucial for the nucleophilic attack on the g-phosphate, is compromised through hydrogen bonding to His-192. It is presumed that, while certain actin residues promote ATP-hydrolysis in Arp2, Arp2 itself might lack the ability to stimulate ATP-hydrolysis within Arp3.

Figure 10. Adapted from Nolen et al. (2004), showing the two rotational axes of the Arp3-subunit: an overlay of the conformation of nucleotide-free Arp3 (red), ADP-bound Arp3 (blue) and ATP-bound Arp3 (cyan): ATP is shown in yellow.

Figure 10. Adapted from Nolen et al. (2004), showing the two rotational axes of the Arp3-subunit: an overlay of the conformation of nucleotide-free Arp3 (red), ADP-bound Arp3 (blue) and ATP-bound Arp3 (cyan): ATP is shown in yellow.

Nucleotides bind to a notch deep within the interface of subdomains 3 and 4 of Arp2; several conserved residues (Gly-306, Ser-307, Met-309) of subdomain 3 form a hydrophobic platform for the bottom of the adenine ring, whereas a tyrosine OH-group (Y310) forms a water-mediated hydrogen bond with the N3 of the adenine ring in ADP. The C-terminal a-helix and a side-chain of Glu-218 form the top of the pocket for the adenine ring and the glutamate-carboxyl group hydrogen-bonds to a conserved arginine in the previous helical turn: a similar interaction is observed in both a- and b-actin. Surprisingly, nucleotide binding did not stabilize the previously found to be instable subdomains 1 and 2; this might explain why additional activator proteins are necessary for actin filament nucleation.

Figure 11. The Arp2/3-dimer with residues essential for ATP-binding and subsequent conformational changes leading to activation of the complex.

Figure 11. The Arp2/3-dimer with residues essential for ATP-binding and subsequent conformational changes leading to activation of the complex.

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