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PDBsum entry 2p9p
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Structural protein
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PDB id
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2p9p
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Contents |
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388 a.a.
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184 a.a.
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341 a.a.
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273 a.a.
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174 a.a.
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165 a.a.
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128 a.a.
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References listed in PDB file
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Key reference
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Title
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Insights into the influence of nucleotides on actin family proteins from seven structures of arp2/3 complex.
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Authors
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B.J.Nolen,
T.D.Pollard.
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Ref.
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Mol Cell, 2007,
26,
449-457.
[DOI no: ]
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PubMed id
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Abstract
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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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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. ADP from 2P9I is yellow, and calcium is green.
(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 cocrystal (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 occur 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.
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Figure 2.
Figure 2. Crosslinking Bovine Arp2/3 Complex Crystals with
Glutaraldehyde Increases Order in Subdomain 1 of Arp2
(A)
Final 3σ F[o] − F[c] electron density map and Cα trace of
modeled regions of Arp2 in uncrosslinked bovine Arp2/3
complex-ATP-Mg^2+ cocrystals (2P9S). The map shows little
density for subdomains 1 and 2.
(B) Final 3σ F[o] − F[c]
omit map and Cσ trace of modeled regions of Arp2 in bovine
Arp2/3 complex-ATP-Ca^2+ 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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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2007,
26,
449-457)
copyright 2007.
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