Critical Conformational Changes in the Arp2/3 Complex Are Induced by Nucleotide and Nucleation Promoting Factor  Erin D. Goley, Stacia E. Rodenbusch, Adam C. Martin, Matthew D. Welch  Molecular Cell  Volume 16, Issue 2, Pages 269-279 (October 2004) DOI: 10.1016/j.molcel.2004.09.018

Figure 1 rArp2/3 CY Complex Is a FRET Probe for Arp2/3 Complex Conformation (A) Illustration of how changes in Arp2/3 complex conformation can be revealed by FRET. Arp3 (dark blue), Arp2 (purple), p40-YFP (yellow), and p21-CFP (light blue) are highlighted. (B) Purified rArp2/3 complexes with CFP and/or YFP tags on p21 and p40 visualized by SDS-PAGE and Coomassie blue staining. Labels: CY = rArp2/3 p21-CFP/p40-YFP, YC = rArp2/3 p21-YFP/p40-CFP, C = rArp2/3 p21-CFP, Y = rArp2/3 p40-YFP. Other labels: XFP = CFP or YFP, FH = Flag and His6 (C) Emission profiles of purified complex(es) after excitation at 435 nm. Labels: CY, YC, C, and Y correspond to the descriptions in (B). Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)

Figure 2 rArp2/3 CY Complex Retains Nucleation and Branching Activities and Supports Listeria Motility In Vitro (A) (Top) Graphs of polymerization of 2 μM actin in the pyrene-actin assay with the indicated concentrations of rArp2/3 CY or untagged rArp2/3 complex and 500 nM GST-WASP-WCA. (Bottom) Calculated concentration of barbed ends generated by increasing concentrations of rArp2/3 CY or untagged complex. (B) Actin filaments polymerized from 4 μM actin, 4 μM rhodamine-phalloidin, 200 nM ActA, and either 10 nM native Arp2/3 complex (nArp2/3) or 1 μM rArp2/3 CY complex. Scale bar = 5 μm. (C) Rhodamine-actin labeled comet tails formed by moving L. monocytogenes (arrowheads) in Xenopus egg extracts that were mock depleted, or depleted of endogenous Arp2/3 complex and supplemented with rArp2/3 CY complex. Scale bar = 10 μm. Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)

Figure 3 Effect of Nucleotide Binding on FRET (A) Box and whiskers plot of FRET/CFP ratios of rArp2/3 CY complex in the absence (con) or presence of MgCl2 (2 mM) and/or ATP/ADP (500 μM). The middle line of each box indicates the median, and the top and bottom lines represent the third and first quartiles. The whiskers indicate the maximum and minimum measurements. (B) Emission profiles of rArp2/3 CY complex in the absence or presence of MgCl2 and/or ATP after excitation at 435 nm. (C) Normalized FRET/CFP ratios of rArp2/3 CY complex over increasing ATP or ADP concentrations and 2 mM MgCl2. (D) Normalized FRET/CFP ratios of rArp2/3 CY complex over increasing ATP concentrations and 2 mM MgCl2 in the presence or absence of GST-WASP-WCA. Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)

Figure 4 Effect of Mutations in the Nucleotide Binding Pockets of Arp2 and Arp3 on Nucleation Activity and FRET (A) (Left) Graphs of polymerization of 2 μM actin over time in the pyrene-actin assay for actin alone (black line) or actin with 500 nM GST-WASP-WCA and 500 nM of the following rArp2/3- CY complexes: wild-type (WT, black circles), Arp3-D11A (3DA, blue triangles), Arp2-D12A (2DA, purple asterisks), Arp3-D11A/Arp2-D12A (2/3DA, green diamonds). (Right) Calculated concentration of barbed ends generated at various concentrations of activated CY complexes. (B) FRET/CFP ratios of wild-type and mutant rArp2/3 CY complexes over increasing Mg-ATP concentrations. (C) (Left) Box and whiskers plot of maximum FRET/CFP ratios of wild-type (black) and mutant rArp2/3 CY (colored) complexes in the absence (con) and presence of 500 μM Mg-ATP. Complexes tested: Arp2-D12A or Arp2-G306Y (2DA or 2GY, purple), Arp3-D11A or Arp3-G324Y (3DA or 3GY, blue), Arp2-D12A/Arp3-D11A or Arp2-G306Y/Arp3-G324Y (2/3DA or 2/3GY, green). (Right) Kds for ATP binding of wild-type and mutant rArp2/3 CY complexes. Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)

Figure 5 Changes in FRET Induced by NPF Binding (A) FRET/CFP ratios of the rArp2/3 CY complex with 500 μM ADP or ATP and 2 mM MgCl2 in the presence or absence of 10 μM GST-WASP-WCA. (B) Normalized FRET/CFP ratio of rArp2/3 CY complex with Mg-ATP/ADP and increasing concentrations of GST-WASP-WCA. (C) Normalized FRET/CFP ratio of rArp2/3 CY complex with Mg-ATP and increasing concentrations of WASP-CA or Scar-CA. (D) FRET/CFP ratio of rArp2/3 CY complex with Mg-ATP and increasing concentrations of cortactin 1–39. (E) FRET/CFP ratios of rArp2/3 CY complex with Mg-ATP or Mg-ATP plus 30 μM GST-WASP-CA or GST-WASP-CA L470A. (F) (left) FRET/CFP ratio of wild-type rArp2/3 CY (wt CY) complex and Arp2-D12A (2DA), or Arp3-D11A (3DA) mutant complexes in the presence or absence of 15 μM GST-WASP-WCA, and (right) calculated Kds for binding to GST-WASP-WCA. Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)

Figure 6 Actin Nucleating Activity of the Arp2/3-W Complex (A) Models for the activity of the rArp2/3 complex tagged on Arp2 with the WH2 (W) domain of WASP (rArp2/3-W). (B) Coomassie-stained SDS-PAGE of purified rArp2/3-W complex. (C) Polymerization of 2 μM actin in the pyrene actin polymerization assay with the indicated concentrations of rArp2/3-W and GST-WASP-WCA (wt or L470A mutant) or GST-WASP-CA (wt or L470A). (D) Polymerization of 2 μM actin in the pyrene actin assay with the indicated concentrations of control rArp2/3 complex and GST-WASP-WCA (wt or L470A) or GST-WASP-CA (wt or L470A). The curve for rArp2/3-W complex and 2 uM GST-WASP-CA from (C) is included for reference. Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)

Figure 7 Model of Conformational Changes Accompanying Arp2/3 Complex Activation (1) Arp2/3 complex devoid of nucleotide binds ATP (T) on Arp2 and/or Arp3, promoting a more compact conformation that facilitates WCA binding. (2) WCA stabilizes a conformation favoring activation and (3) positions actin monomer, leading to (4) nucleation from the side of a mother filament and (5) eventual debranching and recycling of ADP (D) bound Arp2/3 complex. (6) ADP-Arp2/3 exchanges ADP for ATP and repeats the cycle. Stars indicate conditions demonstrated by FRET to change Arp2/3 complex conformation, with the size of the star corresponding to the extent of FRET change. Molecular Cell 2004 16, 269-279DOI: (10.1016/j.molcel.2004.09.018)