Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases

Abstract

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.

Figure 1. Identification of a SPOP Binding Consensus (SBC) sequence in multiple SPOP substrates

(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 Puc^1-390 (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 SPOP^MATH 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 SPOP^MATH 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.

Figure 2. Structural Basis for SPOP^MATH-SBC Interactions

(A) Comparison of 11 independent structures of isolated SPOP^MATH complexed with SBC peptides (3 from Puc – greens; 4 from MacroH2A – cyans/blues; 4 from Ci – pinks/magentas). After superposition over SPOP^MATH mainchain, SBC peptides were displayed with backbones as cartoons and sidechains as sticks, docked in the structure of SPOP^MATH (grey surface) from the complex with Puc^SBC1. The 5 φ-π-S-S/T-S/T motif positions are indicated P1–P5. (B–D) Close-up views of SPOP^MATH (grey) complexes with (B) Puc^SBC1 (green), (C) MacroH2A^SBC (cyan), and (D) Ci^SBC2 (magenta), oriented as in panel A. Dashed lines - hydrogen bonds; red – oxygen; blue – nitrogen; red sphere – water.

Figure 3. Mutational Analysis of SPOP^MATH-Substrate Interaction

(A) BIACORE sensograms showing SPOP^MATH binding to wildtype or indicated mutant versions of a MacroH2A^SBC peptide. Bottom left - Fit used to calculate K_D for SPOP^MATH-MacroH2A^SBC. (B) Representative BIACORE sensograms examining binding between wildtype or indicated mutant versions of SPOP^MATH and a Puc^SBC1 peptide. BIACORE binding data for SPOP^MATH 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.

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 (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 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, I217K mutant SPOP^BTB++Cul3^ntd 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 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, I217K (dimer-defective) mutant SPOP.

Figure 5. A conserved Cul3-Interacting box (3-box)

(A) Structural alignment of SPOP^BTB+, Skp1 (blue) and EloC (yellow). One molecule of the SPOP^BTB+ 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-box^Skp2 (orange) -Cul1 (green) structure (1LDK.pdb), the Elongin C (yellow) – SOCS-box^VHL (magenta) structure (1VCB.pdb) docked onto a structural model of Cul5 (green), and the SPOP^BTB/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 SPOP^BTB (i.e., lacking the 3-box)+Cul3^ntd. 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 SPOP^BTB:Cul3^ntd complex (MW 118.6 kDa) with the indicated K_D 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 SPOP^BTB/3-box and Gigaxonin^BTB/3-box. Residues C-terminal of the Gigaxonin 3-box were omitted for clarity. (F) Superposition of the SPOP and Gigaxonin 3-boxes. SPOP^3-box –cyan; Gigaxonin^3-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.

Figure 6. Crystal Structures of dimeric SPOP^{MATHx-BTB/3-box}-Puc^SBC1

(A) Overall architecture of SPOP^{MATHx-BTB/3-box} dimer. One molecule (A) is colored cyan and the other (B) is red. Each MATH domain binds one Puc^SBC1 (green) peptide. Disordered regions not visible in electron density are represented with dotted lines to show connectivity. (B) Superposition over a BTB domain for SPOP^{MATHx-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 SPOP^{MATH-BTB/3-box} by Endoproteinase Glu-C at room temperature for 30 min. The identities of products determined by Mass Spectrometry are shown. (D) SPOP^MATH and SPOP^BTB/3-box domains do not cofractionate by sizing. A thrombin cleavage site was engineered in the interdomain linker in SPOP^{MATH-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.

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, 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:SPOP^{MATH-BTB/3-box} complex (MW_calc 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 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 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 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.