PDBsum entry 3dwl

Structural protein

PDB id: 3dwl

Contents

Protein chains

355 a.a.

336 a.a. *

272 a.a. *

63 a.a. *

166 a.a. *

114 a.a. *

Ligands

ATP ×2

* Residue conservation analysis

PDB id:

3dwl

Name:

Structural protein

Title:

Crystal structure of fission yeast arp2/3 complex lacking the arp2 subunit

Structure:

Actin-related protein 3. Chain: a, b. Synonym: actin-like protein 3. Actin-related protein 2/3 complex subunit 1. Chain: c, h. Synonym: arp2/3 complex 41 kda subunit, p41-arc. Actin-related protein 2/3 complex subunit 2. Chain: d, i. Synonym: arp2/3 complex 34 kda subunit, p34-arc.

Source:

Schizosaccharomyces pombe. Fission yeast. Organism_taxid: 4896. Strain: tm011. Strain: tm011

Resolution:

3.78Å R-factor: 0.325 R-free: 0.344

Authors:

B.J.Nolen,T.D.Pollard

Key ref:

B.J.Nolen and T.D.Pollard (2008). Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit. J Biol Chem, 283, 26490-26498. PubMed id: 18640983 DOI: 10.1074/jbc.M802607200

Date:

22-Jul-08 Release date: 26-Aug-08

Protein chains

P32390  (ARP3_SCHPO) -  Actin-related protein 3 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 427 a.a.

Struc: 355 a.a.

Protein chains

P78774  (ARPC1_SCHPO) -  Actin-related protein 2/3 complex subunit 1 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 377 a.a.

Struc: 336 a.a.

Protein chains

O14241  (ARPC2_SCHPO) -  Actin-related protein 2/3 complex subunit 2 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 317 a.a.

Struc: 272 a.a.

Protein chains

Q9Y7J4  (ARPC3_SCHPO) -  Actin-related protein 2/3 complex subunit 3 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 174 a.a.

Struc: 63 a.a.

Protein chains

Q92352  (ARPC4_SCHPO) -  Actin-related protein 2/3 complex subunit 4 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 168 a.a.

Struc: 166 a.a.

Protein chains

Q10316  (ARPC5_SCHPO) -  Actin-related protein 2/3 complex subunit 5 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 152 a.a.

Struc: 114 a.a.

DOI no: 10.1074/jbc.M802607200

J Biol Chem 283:26490-26498 (2008)

PubMed id: 18640983

Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit.

B.J.Nolen, T.D.Pollard.

ABSTRACT

Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2Delta Arp2/3 complex) from fission yeast. An x-ray crystal structure of the DeltaArp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2Delta Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2Delta Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

Selected figure(s)

Figure 2.
FIGURE 2. Biochemical characterization of S. pombe Arp2/3 complex with and without Arp2. A, effect of native and Arp2 Arp2/3 complex on the time course of polymerization of pyrene-labeled Mg-ATP actin. Conditions: 4 µM 15% pyrene-labeled chicken skeletal muscle actin, 0.8 µM SpWsp1-VCA, 200 µM complete SpArp2/3 complex ("complete") or Arp2 Arp2/3 complex (" Arp2") in 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl[2], 1 mM EGTA, 0.13 mM ATP, 63 µM CaCl[2], 0.3 mM DTT, 0.6 mM NaN[3] at 22 °C. Thick black line shows 4 µM actin and 0.8 µM SpWsp1-VCA without Arp2/3 complex. Inset shows the concentration of barbed ends when 50% of the actin was polymerized plotted as a function of SpWsp1-VCA concentration for the complete SpArp2/3 complex (pool B). High concentrations of VCA decrease the rate of polymer formation by inhibiting nucleation and slowing pointed end elongation (23, 44). B, equilibrium binding of rhodamine-labeled and unlabeled SpWsp1-VCA to Arp2 Arp2/3 complex and complete Arp2/3 complex measured by fluorescence anisotropy. Conditions: 50 mM KCl, 10 mM imidazole, pH 7.0, 1 mM MgCl[2], 1 mM EGTA, 0.1 mM ATP, 1 mM DTT, and 0.2% thesit. Inset: titration of 100 nM SpWsp1-Rho-VCA with Arp2 (dashed line) and native Arp2/3 complex (solid line). The K[d] values of SpWsp1-Rho-VCA were 120 ± 13 nM for the Arp2 and 49 ± 5 nM for complete Arp2/3 complex. Main plot: titration of 100 nM SpWsp1-Rho-VCA and 300 nM Arp2 (dashed line) or native Arp2/3 complex (solid line) with unlabeled SpWsp1-VCA. Curves were fit as described in the methods giving K[d] values of 0.4 ± 0.1 µM and 0.9 ± 0.1 µM for unlabeled SpWsp1-VCA binding the Arp2 and complete complexes, respectively. C, fluorescence resonance energy transfer to measure binding of SpWsp1-Rho-VCA to OG-actin. Emission scans showing the dependence of the quenching of the fluorescence of 100 nM OG-actin on the concentration of SpWsp1-Rho-VCA in the same buffer as in B. Numbers below the curves indicate Rho-VCA concentrations, in nanomolar. Samples were excited at 480 nm. D, effect of Arp2 Arp2/3 complex and native Arp2/3 complex on binding of OG-actin to Rho-VCA measured by FRET as in C. Plots of fraction of OG-actin emission at 517 nm quenched verses Rho-VCA concentration. Fits of the data gave a K[d] of 15.5 ± 1.7 nM for Rho-VCA binding to OG-actin (solid line, open squares). In the presence of 3 µM native Arp2/3 complex (solid line, filled circles), the K[d] increased to 23.9 ± 1.3 nM. In the presence of 3 µM Arp2-less complex (dashed line, filled triangles) the K[d] was 12.1 ± 1.6 nM.

Figure 4.
FIGURE 4. Stereo figure of electron density of the ARPC1 insert. 2F[o] - F[c] electron density map contoured at 2.0 calculated with phase contributions for the insert region from ARPC1 (subunits C and H, residues 291–311) omitted. C trace of ARPC1 is shown in blue.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 26490-26498) copyright 2008.

Figures were selected by an automated process.