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. 2007 May 11;26(3):449-57.
doi: 10.1016/j.molcel.2007.04.017.

Insights into the influence of nucleotides on actin family proteins from seven structures of Arp2/3 complex

Brad J Nolen  1 , Thomas D Pollard
Affiliations

Insights into the influence of nucleotides on actin family proteins from seven structures of Arp2/3 complex

Brad J Nolen et al. Mol Cell. .

Abstract

ATP is required for nucleation of actin filament branches by Arp2/3 complex, but the influence of ATP binding and hydrolysis are poorly understood. We determined crystal structures of bovine Arp2/3 complex cocrystallized with various bound adenine nucleotides and cations. Nucleotide binding favors closure of the nucleotide-binding cleft of Arp3, but no large-scale conformational changes in the complex. Thus, ATP binding does not directly activate Arp2/3 complex but is part of a network of interactions that contribute to nucleation. We compared nucleotide-induced conformational changes of residues lining the cleft in Arp3 and actin structures to construct a movie depicting the proposed ATPase cycle for the actin family. Chemical crosslinking stabilized subdomain 1 of Arp2, revealing new electron density for 69 residues in this subdomain. Steric clashes with Arp3 appear to be responsible for intrinsic disorder of subdomains 1 and 2 of Arp2 in inactive Arp2/3 complex.

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Figures

Figure 1
Figure 1
Nucleotide binding causes changes in the cleft of Arp3. (A) Superposition of Cα traces of apo-Arp2/3 complex (1K8K) and the ADP-cocrystallized, crosslinked Arp2/3 complex (2P9I). Structures were superposed by aligning subdomains 1 and 2 of Arp3. ARPC1, ARPC2, ARPC4, ARPC5 and Arp2 overlay well (both complexes gray). A rigid body motion of subdomains 3 and 4 of Arp3 and ARPC3 (cyan in ADP-complex, red in apo-complex) closes the cleft of structure 2P9I. Additional residues built for subdomain 1 in Arp2 of the crosslinked ADP cocrystals (2P9I) are also in cyan. (B) Stereo figure showing overlay of the nucleotide binding cleft of Arp3 in the crosslinked ADP cocrystal (2P9I, cyan) and the crosslinked ATP cocrystal. (2P9K, yellow). ADP is magenta and ATP is purple. Labeled residues mark key features: Thr14 for the P1 loop; Val174 for the P2 loop; and His80 for the sensor loop. Two distances define the width of the cleft: B1 (Thr14 Cα to Gly173 Cα; atoms shown as orange spheres) and B2 (Gly15 Cα to Asp172 Cα; atoms shown as brown spheres). Distance B2 was used to categorize clefts in structures of Arp2, Arp3 and actin as open, closed or intermediate (Table 2). (C) Stereo figure showing an overlay of the nucleotide binding cleft of Arp3 in the crosslinked ADP co-crystal (2P9I, cyan protein, magenta ADP) and the previously published ADP-soaked structure (1U2V, orange protein, yellow ADP). (D) Summary of conformational changes in the ATP binding cleft during the ATPase cycle of actin family proteins. The dotted lines show hydrogen bonds between the γ-phosphate and the loops when the cleft is fully closed. Residue numbering is for bovine Arp3. Conformational changes observed in Arp3 and actin are numbered in red. Wavy red lines connect structural features that show correlated changes in one or more actin or Arp3 structures. (1) Two positions of a valine (Val174 in Arp3) in the P2 loop observed in Arp3 and actin structures suggest this valine may be involved in sensing the nucleotide binding state. (2) A rotomer flip in Ser14 (Thr14 in Arp3) and a slight inward collapse (cyan arrowhead) of the P1 loop occurs in the ADP-actin structures (1J6Z and 2HF4) and in a structure of Arp3 with bound ADP (2P9I). This movement is accompanied by a flip of the backbone carbonyl of a residue in the sensor loop in both Arp3 and actin. (3) Rigid body motions of subdomains 1 and 2 relative to subdomains 3 and 4 result in opening or closing of the nucleotide cleft. These structural changes have been observed in actin by comparing the single open structure (1HLU) to each of the other ADP and ATP-containing structures, all of which are closed. Open and closed conformations have also been observed in Arp3, where the nucleotide state is correlated to the degree of opening of the cleft.
Figure 2
Figure 2
Crosslinking bovine Arp2/3 complex crystals with glutaraldehyde increases order in subdomain 1 of Arp2. (A) Final 3σ Fo-Fc electron density map and Cα trace of modeled regions of Arp2 in uncrosslinked bovine Arp2/3 complex-ATP-Mg2+ cocrystals (2P9S). The map shows little density for subdomains 1 and 2. (B) Final 3σ Fo-Fc omit map and Cσ trace of modeled regions of Arp2 in bovine Arp2/3 complex-ATP-Ca2+ cocrystals treated with glutaraldehyde (2P9K). The newly modeled regions were not included in the map calculation. (C) Steric hindrance with Arp3 may prevent Arp2 from closing in the inactive complex. Cα traces show Arp3 (cyan) and Arp2 (blue) from the structure of crosslinked ADP-Arp2/3 complex (2P9I) with the addition of a model of four disordered residues at the end of the αK/β15 loop of Arp3 (orange). Actin (red) is overlaid onto Arp2 to show potential clashes (red arrows) of subdomain 2 with the αI/αJ loop and the αK/β15 loop of Arp3. The yellow Cα trace (highlighted with arrow) shows how αK is connected to β15 in actin. The αK/β15 insert in Arp3 makes the αK helix three turns longer in Arp3 than actin.
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