1. Volume 36, Issue 1, Pages 39-50 (October 2009)
Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB- Cul3 Ubiquitin Ligases 
Min Zhuang, Matthew F. Calabrese, Jiang Liu, M. Brett Waddell, Amanda Nourse, Michal Hammel, Darcie J. Miller, Helen Walden, David M. Duda, Steven N. Seyedin, Timothy Hoggard, J. Wade Harper, Kevin P. White, Brenda A. Schulman 
Molecular Cell 
Volume 36, Issue 1, Pages (October 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of an SBC Sequence in Multiple SPOP Substrates
(A) Identification of a SPOP-binding peptide in Puc. Left, Coomassie-stained SDS-PAGE gel showing products after trypsin digestion of a complex between a SPOP MATH domain and Puc1–390 (333:1 trypsin, 3 hr at RT). Right, Coomassie-stained SDS-PAGE gel of fractions from gel filtration (SD200) of trypsin digest products. Bottom, peptide copurifying with SPOPMATH in fractions 33–35, identified by mass spectrometry.
(B) Identification of a MacroH2A sequence required for binding to SPOP. Top, schematic view of MacroH2A deletions, highlighting residues 166–179. Bottom, Coomassie-stained SDS-PAGE gel of GST pull-downs of GST, GST-MacroH2AΔ1, and GST-MacroH2AΔ2 coexpressed in E. coli with a His-MBP-tagged SPOP MATH domain.
(C) SBCs (red) in SPOP substrates Puc, MacroH2A, Ci, and Daxx.
(D) Binding constants for SPOPMATH interactions with SBC peptides, measured by surface plasmon resonance (Biacore3000). Error represents standard error from five concentrations measured in triplicate.
(E) Roles of individual Puc SBCs in in vitro ubiquitination. Western blots detecting His-Puc ubiquitination for wild-type (WT) and mutant Puc substrates. SBCm1, SBCm2, and SBCm3 refer to mutation at the three SBC sites.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2 Structural Basis for SPOPMATH-SBC Interactions
(A) Comparison of 11 independent structures of isolated SPOPMATH complexed with SBC peptides (3 from Puc, greens; 4 from MacroH2A, cyans/blues; 4 from Ci, pinks/magentas). After superposition over SPOPMATH main chain, SBC peptides were displayed with backbones as cartoons and side chains as sticks, docked in the structure of SPOPMATH (gray surface) from the complex with PucSBC1. The five ϕ-π-S-S/T-S/T motif positions are indicated by P1–P5.
(B–D) Close-up views of SPOPMATH (gray) complexes with PucSBC1 (green) (B), MacroH2ASBC (cyan) (C), and CiSBC2 (magenta) (D), oriented as in (A). Dashed lines, hydrogen bonds; red, oxygen; blue, nitrogen; red sphere, water.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3 Mutational Analysis of SPOPMATH-Substrate Interaction
(A) Biacore sensograms showing SPOPMATH binding to wild-type or indicated mutant versions of a MacroH2ASBC peptide. Bottom left, fit used to calculate KD for SPOPMATH-MacroH2ASBC.
(B) Representative Biacore sensograms examining binding between wild-type or indicated mutant versions of SPOPMATH and a PucSBC1 peptide. Biacore binding data for SPOPMATH mutants and SBC peptides are summarized below. Error represents standard error from five concentrations measured in triplicate.
(C) Western blots showing association of HA-SPOP (top) with Myc-Puc (bottom) or mutants after anti-Myc immunoprecipitation from Drosophila S2 cells cotransfected with the indicated constructs. D130 of human SPOP corresponds to D159 in Drosophila SPOP, W131 corresponds to W160, and Y87 corresponds to Y116. ∗ indicates processed form of Puc (Liu et al., 2009).
(D) Anti-Myc western blot detecting ubiquitination of Puc from S2 cells cotransfected with wild-type or mutant versions of SPOP.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4 SPOPBTB+ Forms a 2:2 Dimer with Cul3 N-Terminal Domain
(A) Left, overall view of the SPOPBTB+ dimer, with protomers in cyan (A) and red (B). Right, close-up view of dimer interface rotated 90° in x.
(B) Equilibrium AUC of SPOPBTB+ + Cul3NTD. 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 SPOPBTB+:Cul3NTD complex (MW kDa) with indicated KD value. For clarity, only the 3 μM sample is shown.
(C) AUC of L186D, L190D, L193D, and I217K mutant SPOPBTB+ + Cul3NTD 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 SPOPBTB+ (mutant):Cul3NTD complex (MW 63.6 kDa) with the indicated KD value. For clarity, only the 2.0 μM sample is shown.
(D) Western blots of SPOPMATH-BTB+-mediated ubiquitination detecting His-Puc for wild-type and L186D, L190D, L193D, and I217K (dimer-defective) mutant SPOP.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5 A Conserved 3-Box
(A) Structural alignment of SPOPBTB+, Skp1 (blue), and EloC (yellow). One molecule of the SPOPBTB+ dimer is cyan (SPOP_A) and one is red (SPOP_B). The SPOP helix pair (above “+”) not shared in Skp1 and EloC is labeled “3-box.”
(B) Structural comparison of the Skp1-F-boxSkp2-Cul1 structure (colored blue/orange/green; 1LDK.pdb), with the EloC-SOCS-boxVHL structure (1VCB.pdb) docked onto a structural model of Cul5 (yellow/magenta/green) and the SPOPBTB/3-box structure docked on a structural model of Cul3 (cyan/green). The relative locations of the SPOP 3-box, F-box, and SOCS-box are indicated.
(C) Equilibrium AUC of SPOPBTB (i.e., lacking the 3-box) + Cul3NTD. Samples at 1.2–8.0 μM centrifuged at 8,000 (red), 12,000 (blue), and 15,000 (black) rpm (4°C). Lines represent the global nonlinear least-squares best-fit of all data sets/concentrations/speeds to a heterogeneous association model describing a 2:2 SPOPBTB:Cul3NTD complex (MW kDa) with the indicated KD value. For clarity, only the 3.0 μM sample is shown.
(D) Schematic of SPOP and Gigaxonin (Gig) domain arrangements, which represent two distinct BTB subfamilies, MATH-BTB and BTB-Kelch, respectively.
(E) Overall structural alignment of SPOPBTB/3-box and GigBTB/3-box. Residues C-terminal of the Gig 3-box were omitted for clarity.
(F) Superposition of the SPOP and Gig 3-boxes. SPOP3-box, cyan; Gig3-box, light blue.
(G) Structure based sequence alignment (ESPript) of 32 amino acids corresponding to 3-boxes from five Cul3-interacting BTB proteins. SPOP 3-box residue numbers are shown on top.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6 Crystal Structures of Dimeric SPOPMATHx-BTB/3-box-PucSBC1
(A) Overall architecture of SPOPMATHx-BTB/3-box dimer. One molecule (A) is colored cyan and the other (B) is red. Each MATH domain binds one PucSBC1 (green) peptide. Disordered regions not visible in electron density are represented with dotted lines to show connectivity.
(B) Superposition over a BTB domain for SPOPMATHx-BTB/3-box structures determined from crystals with slightly different unit cells. MATH_A1 (cyan) and MATH_B1 (red) are from crystal form 1 and correspond to the structure in (A). MATH_A2 (orange) and MATH_B2 (blue) are from crystal form 2.
(C) Coomassie-stained SDS-PAGE gel showing products of proteolysis of SPOPMATH-BTB/3-box by endoproteinase Glu-C at RT for 30 min. The identities of products determined by mass spectrometry are shown.
(D) SPOPMATH and SPOPBTB/3-box domains do not cofractionate by sizing. A thrombin cleavage site was engineered in the interdomain linker in SPOPMATH-BTB/3-box (−T), and after treatment with thrombin (+T), the product was subject to gel filtration chromatography. Individual fractions were analyzed by Coomassie-stained SDS-PAGE gel, below.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7 A 1:2 Substrate Complex with the SPOP-Cul3 Ubiquitin Ligase
(A) Velocity AUC of SPOPMATH-BTB/3-box + Puc1–390 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:SPOPMATH-BTB/3-box complex (MWcalc of kDa) and excess free Puc (MWcalc 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 SPOPMATH-BTB/3-box:Puc + Puc. For clarity, only the 1.1 μM sample is shown.
(C) Overall structure of SPOPMATHx-MacroH2ASBC (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.
Molecular Cell  , 39-50DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions