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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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