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1.
Figure 2

Figure 2. Structural Basis for SPOPMATH-SBC Interactions. From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(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 mainchain, SBC peptides were displayed with backbones as cartoons and sidechains as sticks, docked in the structure of SPOPMATH (grey surface) from the complex with PucSBC1. The 5 φ-π-S-S/T-S/T motif positions are indicated P1–P5.
(B–D) Close-up views of SPOPMATH (grey) complexes with (B) PucSBC1 (green), (C) MacroH2ASBC (cyan), and (D) CiSBC2 (magenta), oriented as in panel A. Dashed lines - hydrogen bonds; red – oxygen; blue – nitrogen; red sphere – water.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.
2.
Figure 3

Figure 3. Mutational Analysis of SPOPMATH-Substrate Interaction. From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(A) BIACORE sensograms showing SPOPMATH binding to wildtype 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 wildtype or indicated mutant versions of SPOPMATH and a PucSBC1 peptide. BIACORE binding data for SPOPMATH mutants and SBC peptides is summarized below.
(C) Western blots showing association of HA-SPOP (top) with Myc-Puc (bottom) or mutants after anti-Myc immunoprecipitation from Drosophila S2 cells co-transfected with the indicated constructs. D130 of human SPOP corresponds to D159 in Drosophila SPOP, W131 corresponds to W160, and Y87 corresponds to Y116. *Processed form of Puc (Liu et al., 2009).
(D) Anti-Myc western blot detecting ubiquitination of Puc from S2 cells co-transfected with wild-type or mutant versions of SPOP.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.
3.
Figure 4

Figure 4. SPOPBTB+ forms a 2:2 dimer with Cul3 N-terminal domain. From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(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 (red), 12 (blue), and 16 (black) krpm 4 C. Lines show global nonlinear least-squares best-fit of all datasets/concentrations/speeds to a heterogeneous association model describing a 2:2 SPOPBTB+:Cul3ntd complex (MW 127.1 kDa) with indicated KD value. For clarity, only the 3 μM sample is shown.
(C) AUC of L186D, L190D, L193D, I217K mutant SPOPBTB++Cul3ntd performed as in B. Lines show global nonlinear least-squares best-fit of all datasets/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, I217K (dimer-defective) mutant SPOP.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.
4.
Figure 1

Figure 1. Identification of a SPOP Binding Consensus (SBC) sequence in multiple SPOP substrates. From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(A) Identification of a SPOP-binding peptide in Puc. Left, Coommassie-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, rt). Right, Coommassie-stained SDS-PAGE gel of fractions from gel filtration (SD200) of trypsin digest products. Bottom, peptide co-purifying 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, Coommassie-stained SDS-PAGE gel of GST-pull-downs of GST, GST-MacroH2A 1, and GST-MacroH2A 2 coexpressed in E. coli with a HisMBP-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).
(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 3 SBC sites.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.
5.
Figure 6

Figure 6. Crystal Structures of dimeric SPOPMATHx-BTB/3-box-PucSBC1. From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(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) Coommassie-stained SDS-PAGE gel showing products of proteolysis of SPOPMATH-BTB/3-box by Endoproteinase Glu-C at room temperature 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 Coommassie-stained SDS-PAGE gel, below.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.
6.
Figure 5

Figure 5. A conserved Cul3-Interacting box (3-box). From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(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 (blue) -F-boxSkp2 (orange) -Cul1 (green) structure (1LDK.pdb), the Elongin C (yellow) – SOCS-boxVHL (magenta) structure (1VCB.pdb) docked onto a structural model of Cul5 (green), and the SPOPBTB/3-box (cyan) docked on a structural model of Cul3 (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 to 8.0 μM centrifuged at 8 (red), 12 (blue), and 15 (black) krpm 4 C. Lines represent the global nonlinear least-squares best-fit of all datasets/concentrations/speeds to a heterogeneous association model describing a 2:2 SPOPBTB:Cul3ntd complex (MW 118.6 kDa) with the indicated KD value. For clarity, only the 3.0 μM sample is shown.
(D) Schematic of SPOP and Gigaxonin domain arrangements, which represent two distinct BTB subfamilies, MATH-BTB and BTB-Kelch, respectively.
(E) Overall structural alignment of SPOPBTB/3-box and GigaxoninBTB/3-box. Residues C-terminal of the Gigaxonin 3-box were omitted for clarity.
(F) Superposition of the SPOP and Gigaxonin 3-boxes. SPOP3-box –cyan; Gigaxonin3-box - light blue.
(G) Structure based sequence alignment (ESPript) of 32 amino acids corresponding to 3-boxes from 5 Cul3-interacting BTB proteins. SPOP 3-box residue numbers are shown on top.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.
7.
Figure 7

Figure 7. A 1:2 substrate complex with the SPOP-Cul3 ubiquitin ligase. From: Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases.

(A) Velocity AUC of SPOPMATH-BTB/3-box + Puc1-390 at 20 °C, 60 krpm 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 112.5 kDa) and excess free Puc (MWcalc of 42.1 kDa).
(B) Equilibrium AUC of a sample as in (A). Samples at 1 to 6 μM centrifuged at 8 (red), 12 (blue), and 16 (black) krpm 4 C. Lines show global nonlinear least squares best-fit of all datasets/concentrations/speeds to a heterogeneous association model with 2 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 grey, 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.

Min Zhuang, et al. Mol Cell. ;36(1):39-50.

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