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Cullin-Ring ubiquitin ligases (CRLs) are E3 complexes that specifically recognize substrates through substrate adaptors. In the largest CRL subfamily, Cul3 binds a BTB domain, and a protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH and BTB domain containing protein, SPOP, which regulates diverse signaling pathways. First, we identified a conserved SPOP Binding Consensus (SBC) motif in the transcriptional regulator Ci, the protein phosphatase Puc, and the chromatin component MacroH2A. The SBC motif specifically binds the MATH domain of SPOP, and is required for Puc ubiquitination in vitro and in vivo. Structural determination and analysis of SPOPMATH in complex with peptides encompassing the SBC motif revealed the molecular basis for recognition of diverse substrates by SPOP. Second, the dimeric BTB domain of SPOP assembles into a dimer with Cul3, an interaction that is facilitated by a helical motif that we term 3-box due to resemblance to F- and SOCS-boxes in other cullin-based E3s. 3-box is also found in the BTB protein Gigaxonin and is predicted in a subset of BTB proteins. Furthermore, structures of nearly full-length SPOP constructs indicate flexibility between the MATH and BTB domains, potentially allowing regulation of diverse substrates. Those such as Puc with multiple SBCs may functionally interact with a single SPOP dimer. All together, this study provides a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3, and how their dimerization and structural variability may facilitate recognition and ubiquitination of diverse substrates.