Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases

Abstract

SUMMARY 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.

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…Although the BACK domain (Fig 1C) has been crystallized as a dimer (van Geersdaele et al , 2013), oligomeric states ranging from dimers to tetramers or pentamers have been proposed (Errington et al , 2012; van Geersdaele et al , 2013). Truncated SPOP constructs encoding the BTB and BACK domains self‐associate into higher‐order oligomers (Errington et al , 2012) that possess increased ubiquitination efficiency, supporting the functional importance of oligomerization (Zhuang et al , 2009; Errington et al , 2012). Mutations within the MATH domain perturb interactions with substrates (Geng et al , 2013, 2014; Theurillat et al , 2014; Zeng et al , 2014; Zhang et al , 2015).…”

“…SPOP is a substrate adaptor of a cullin‐3‐RING ubiquitin ligase (CRL3) and serves to recruit substrates to the CRL3 (Zhuang et al , 2009) for subsequent ubiquitination and degradation (Hernández‐Muñoz et al , 2005; Kent et al , 2006; Kwon et al , 2006; Zhang et al , 2006; Li  et al , 2008). The Drosophila melanogaster homolog of SPOP, Roadkill/HIB, is essential for early development (Kent et al , 2006).…”

“…Recent efforts to sequence the cancer genome have identified many mutations in SPOP that are associated with cancers (Lawrence et al , 2014), notably prostate (Kim et al , 2013), breast (Kim et al , 2011), endometrial (Le Gallo et al , 2012), and gastric (Kim et al , 2013) cancers [Appendix Fig S1 and www.cbioportal.org (Cerami et al , 2012; Gao et al , 2013)]. SPOP contains three domains; it recruits substrates through its MATH ( m eprin a nd t raf h omology) domain (Zhuang et al , 2009), forms dimers through its BTB ( b ric à brac, t ramtrack, b road complex) domain (Zhuang et al , 2009; Errington et al , 2012), and forms either dimers or tetramers through its BACK ( B TB a nd C ‐terminal K elch) domain (Errington et al , 2012; van Geersdaele et al , 2013 and Fig 1A). The BTB dimer interface (Fig 1B) has been well characterized in crystal structures of truncated versions of SPOP (Zhuang et al , 2009; Errington et al , 2012).…”

“…SPOP contains three domains; it recruits substrates through its MATH ( m eprin a nd t raf h omology) domain (Zhuang et al , 2009), forms dimers through its BTB ( b ric à brac, t ramtrack, b road complex) domain (Zhuang et al , 2009; Errington et al , 2012), and forms either dimers or tetramers through its BACK ( B TB a nd C ‐terminal K elch) domain (Errington et al , 2012; van Geersdaele et al , 2013 and Fig 1A). The BTB dimer interface (Fig 1B) has been well characterized in crystal structures of truncated versions of SPOP (Zhuang et al , 2009; Errington et al , 2012). Although the BACK domain (Fig 1C) has been crystallized as a dimer (van Geersdaele et al , 2013), oligomeric states ranging from dimers to tetramers or pentamers have been proposed (Errington et al , 2012; van Geersdaele et al , 2013).…”

“…The previously described mutation Y353E (van Geersdaele et al , 2013) was introduced into the BACK domain so that self‐association could only occur through BTB ‐mediated interactions resulting in SPOP mut BACK ; previously described mutations of the conserved BTB interface (Zhuang et al , 2009) resulted in SPOP mut BTB , which self‐associates only through BACK ‐mediated interactions. Constructs for expression in mammalian cells encode the full‐length protein; those for expression in bacteria comprise residues 28–359.…”