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.