Structural complexity in the KCTD family of Cullin3-dependent E3 ubiquitin ligases.

Daniel M Pinkas,Fiona J Sorrell,Joshua C Bufton,Caroline E Sanvitale,James Doutch,Nicolae Solcan,Rod Chalk,Alex N Bullock

doi:10.1042/bcj20170527

Daniel M Pinkas, Fiona J Sorrell + Show 6 more

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https://doi.org/10.1042/bcj20170527

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Abstract

Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB–BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.

Highlights

The human potassium Channel Tetramerization Domain (KCTD) family contains 25 soluble proteins that share a conserved potassium (K+) Channel Tetramerization Domain at their N-termini and have variable C-termini [1]
A further unexpected structure is observed for the BTB domain of SHKBP1, which adopts a monomeric state in the absence of its binding partner Cul3 (Figure 1B)
We present X-ray crystal structures of five novel BTB domains from the KCTD family that reveal previously unobserved oligomeric forms and geometries

Summary

Introduction

The human KCTD family contains 25 soluble proteins that share a conserved potassium (K+) Channel Tetramerization Domain (a subtype of ‘BTB domain’) at their N-termini and have variable C-termini [1]. The most common BTB domain hetero-protein interaction is with the N-terminal domain of the cullin family protein Cul, which recruits specific BTB-containing proteins into Cullin-RING E3 ligase (CRL3) complexes (reviewed in [4,5,6,7,8]) In this manner, the core BTB domain is structurally and functionally analogous to the substrate adaptors Skp and Elongin C, which bind to Cul and Cul2/5, respectively [9,10,11]. KCTD17 regulates ciliogenesis by polyubiquitylating trichoplein [19] and its missense mutation is associated with autosomal-dominant myoclonus-dystonia [20] Mutations in another KCTD family member SHKBP1 [(SH3-Domain Kinase Binding Protein 1)-Binding Protein 1] have been identified in cervical cancer [21] and acute myeloid leukaemia [22]. Cul is able to induce the reassembly of a subset of these KCTD family members into 5 : 5 heterodecamers establishing a conserved architecture for these E3 ligase complexes

Results

Discussion

Experimental procedures