Adaptor Protein Self-Assembly Drives the Control of a Cullin-RING Ubiquitin Ligase

Abstract

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

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

“…Some of these assemblies form via nucleation‐driven polymerization and result in stable complexes, and we are starting to understand the structural basis for their proximity‐enhanced activation (Korennykh et al , 2009; Yin et al , 2009; Li et al , 2012a; Lu et al , 2014). Other assemblies depend on transient interactions and are more labile, generating oligomers with broad size distributions (Rivas et al , 2000; Errington et al , 2012; Canzio et al , 2013). The functions of these more labile higher‐order oligomers are not well understood.…”

“…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).…”

“…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).…”

“…The domains are colored as in (A) ( MATH , green; BTB , red; and BACK , blue), with one monomer shown in gray for clarity. Introduction of these mutations does not abolish the interaction between SPOP and the CRL because they are not part of the SPOP /cullin‐3 interface (Errington et al , 2012; Zhuang et al , 2009) and because mutation of the BTB domain or deletion of the BACK domain does not prevent SPOP from promoting ubiquitination of substrates (Zhuang et al , 2009; Errington et al , 2012). …”