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PDBsum entry 3hqi
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Protein binding, ligase
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PDB id
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3hqi
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References listed in PDB file
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Key reference
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Title
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Structures of spop-Substrate complexes: insights into molecular architectures of btb-Cul3 ubiquitin ligases.
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Authors
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M.Zhuang,
M.F.Calabrese,
J.Liu,
M.B.Waddell,
A.Nourse,
M.Hammel,
D.J.Miller,
H.Walden,
D.M.Duda,
S.N.Seyedin,
T.Hoggard,
J.W.Harper,
K.P.White,
B.A.Schulman.
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Ref.
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Mol Cell, 2009,
36,
39-50.
[DOI no: ]
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PubMed id
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Abstract
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In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated
protein-interaction domain such as MATH recruits substrates for ubiquitination.
Here, we present biochemical and structural analyses of the MATH-BTB protein,
SPOP. We define a SPOP-binding consensus (SBC) and determine structures
revealing recognition of SBCs from the phosphatase Puc, the transcriptional
regulator Ci, and the chromatin component MacroH2A. We identify a dimeric
SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB
domains, which we call "3-box" due to its facilitating Cul3 binding and its
resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility
between the substrate-binding MATH and Cul3-binding BTB/3-box domains
potentially allows a SPOP dimer to engage multiple SBCs found within a single
substrate, such as Puc. These studies provide a molecular understanding of how
MATH-BTB proteins recruit substrates to Cul3 and how their dimerization and
conformational variability may facilitate avid interactions with diverse
substrates.
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Figure 4.
Figure 4. SPOP^BTB+ Forms a 2:2 Dimer with Cul3 N-Terminal
Domain
(A) Left, overall view of the SPOP^BTB+ dimer, with
protomers in cyan (A) and red (B). Right, close-up view of dimer
interface rotated 90° in x.
(B) Equilibrium AUC of
SPOP^BTB+ + Cul3^NTD. Samples at 1.0 to 8.8 μM centrifuged at
8,000 (red), 12,000 (blue), and 16,000 (black) rpm (4°C).
Lines show global nonlinear least-squares best-fit of all data
sets/concentrations/speeds to a heterogeneous association model
describing a 2:2 SPOP^BTB+:Cul3^NTD complex (MW 127.1 kDa) with
indicated K[D] value. For clarity, only the 3 μM sample is
shown.
(C) AUC of L186D, L190D, L193D, and I217K mutant
SPOP^BTB+ + Cul3^NTD performed as in (B). Lines show global
nonlinear least-squares best-fit of all data
sets/concentrations/ speeds to a heterogeneous association model
describing a 1:1 SPOP^BTB+ (mutant):Cul3^NTD complex (MW 63.6
kDa) with the indicated K[D] value. For clarity, only the 2.0
μM sample is shown.
(D) Western blots of
SPOP^MATH-BTB+-mediated ubiquitination detecting His-Puc for
wild-type and L186D, L190D, L193D, and I217K (dimer-defective)
mutant SPOP.
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Figure 7.
Figure 7. A 1:2 Substrate Complex with the SPOP-Cul3
Ubiquitin Ligase
(A) Velocity AUC of SPOP^MATH-BTB/3-box +
Puc^1–390 at 20°C and 60,000 rpm fit to a continuous
distribution model c(s). Two peaks indicate molecular weights of
110 kDa and 39 kDa, corresponding to the 1:2
Puc:SPOP^MATH-BTB/3-box complex (MW[calc] of 112.5 kDa) and
excess free Puc (MW[calc] of 42.1 kDa).
(B) Equilibrium AUC
of a sample as in (A). Samples at 1–6 μM centrifuged at 8,000
(red), 12,000 (blue), and 16,000 (black) rpm (4°C). Lines
show global nonlinear least-squares best-fit of all data
sets/concentrations/speeds to a heterogeneous association model
with two species, 2:1 SPOP^MATH-BTB/3-box:Puc + Puc. For
clarity, only the 1.1 μM sample is shown.
(C) Overall
structure of SPOP^MATHx-MacroH2A^SBC (pep2). Two isolated MATH
domains (chain A, cyan; chain B, pink) bind a single-substrate
peptide (green) at two suboptimal SBC sites.
(D) Schematic
view of a SPOP-Cul3 ubiquitin ligase bound to a single
substrate. Substrate is shown in gray, with SBCs in green and
ubiquitin-acceptor lysines as Ks. The two protomers of the
dimeric SPOP complex are shown in cyan and red, with each
BTB/3-box bound near the N terminus of an elongated Cul3 (olive)
activated with NEDD8 (orange) near the C terminus. E2-bound Rbx1
RING domains are shown flexibly tethered to the Cul3 C-terminal
domains. The high degree of conformational flexibility may allow
substrates with a range of SBC configurations to be
polyubiquitinated at multiple sites.
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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2009,
36,
39-50)
copyright 2009.
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