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

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.

Keywords: BTB; Cul3; Cullin-RING ligase; crystallography; protein–protein interaction; ubiquitination.

Figure 1.. Overview of the BTB domain structures.

(A) Previously reported X-ray structure of the four-fold rotationally symmetric tetramer of the BTB domain of human potassium channel Kv4.3 (PDB ID 1S1G). (B) Novel X-ray structures reported here: Monomer – SHKBP1 (4CRH); two-fold rotationally symmetric tetramers – KCTD10 (5FTA) and KCTD13 (4UIJ); C-shaped pentamer – KCTD16 (5A15); and closed pentamer – KCTD17 (5A6R). (C) Phylogenetic tree of the KCTD family BTB domains. The previously reported structures of KCTD1, KCTD5 and KCTD9 are highlighted in tan, and the novel structures reported here are highlighted in green. (D) Sequence alignment of selected BTB domains.

Figure 2.. Interfaces mediating oligomer formation.

(A) Ribbon representation of a KCTD10 monomer coloured from N-terminus (blue) to C-terminus (red). (B) KCTD10 tetramer with strands β1 and β2 highlighted. Top – the common interface in which the β1–β2 hairpin packs end-on to its neighbour. Bottom – in the secondary interface observed only in KCTD10 and KCTD13, the β2 strands on neighbouring molecules pack in an antiparallel fashion. The two-fold rotational axis runs directly into the page between the highlighted β hairpins. (C) The BTB domains of Kv4.3 (blue) and KCTD16 (red) are superimposed to indicate their different geometries at the common interface (β1–β2 hairpins are highlighted). (D) Ribbon representation of KCTD10 with residues at the secondary interface shown highlighted as sticks. (E) Surface representation of KCTD10 coloured by sequence conservation (green for most conserved and yellow for least conserved). High sequence conservation across the KCTD family is apparent at the common interface (top – as observed in the X-ray structure; bottom – BTB domains are rotated 90° with respect to the X-ray structure. (F) Side chains in the common interface between two KCTD10 BTB domains are shown and coloured as in panel E top. Thick and thin sticks highlight the residues from the BTBs on the left and right, respectively. Hydrogen bonds are shown as dashed lines with bonds across the interface shown in red and within a BTB in grey. (G) The icosahedral KCTD17 60-mer is shown as a cartoon with cylindrical helices. On the left, the pentamer mediated by the common interface is highlighted in orange. On the right, BTBs involved in the secondary interface are highlighted in orange.

Figure 3.. Solution states of BTB domains.

(A) Small angle X-ray scattering of indicated BTB domain assemblies. Data are shown in green and scattering calculated from the particles observed in the X-ray structures shown as black lines. Upper left – scattering calculated for the KCTD10 tetramer (5FTA, solid black line) is compared with scattering calculated for the distinct structure of Kv 4.3 (1S1G, dashed black line). Chi value for SAXS fit – 2.2 asymmetric tetramer, 0.8 symmetric tetramer. Upper right – scattering calculated for the KCTD16 C-shaped pentamer (5A15, solid black line) is compared with scattering calculated for the closed KCTD5 pentamer (3DRZ, dashed black line). Chi value for SAXS fit – 1.8 open pentamer, 0.8-closed pentamer. Lower left – scattering calculated for the icosahedral structure of 6His-KCTD17 is shown (5A6R, solid black line). Reduced Chi square for SAXS fit – 0.53. A red cross denotes that this assembly was not observed. (B) Native mass spectrum of 6His-KCTD17. Charge states corresponding to KCTD17 60-mer ions (neutral mass 920 856 Da) are indicated. A charge radius value of 85.5 Å was calculated from independent MS analyses using +79 as the most abundant ion and the method of Testa et al. [32]. The calculated value of 85.5 Å was in excellent agreement with the observed radius in the crystal structure of 84 Å. The expected mass of the complex is the observed monomer mass (15347.7 Da) multiplied by 60 = 920 862 Da. The observed mass of the complex is m/z 11699.4 × 79 − 79 = 924173.6 Da. This corresponds to an observed mass difference of 0.35%. (C) Results of SEC–MALS. Left – UV (280 nm) traces of BTB domains. Retention volumes corresponding to peaks are indicated. Right – masses derived from multi-angle laser light scattering measurements are compared with masses calculated from X-ray structures.

Figure 4.. Determinants of KCTD17 60-mer stability.

(A) Size-exclusion traces (UV 280 nm) of 6xHis-KCTD17^BTB under various running buffer conditions (full buffer conditions described in methods). (B) Homology model of multi-domain KCTD17 based on the known structure of KCTD5 [29]. (C) Size-exclusion traces (UV 280 nm) of indicated 6xHis-tagged KCTD17 constructs. (D) Size-exclusion traces (UV 280 nm) of indicated tag-cleaved KCTD17 constructs.

Figure 5.. ITC measurements of Cul3 binding to KCTD family proteins.

(A) Data for selected isolated BTB domains. (B) Comparison of single and multi-domain constructs of KCTD17. (C) Comparison of single- and multi-domain constructs of KCTD5.

Figure 6.. KCTD family BTB domains bind to Cul3 as 5 : 5 heterodecamers.

Left panels – size-exclusion chromatograms (UV 280 nm) of indicated KCTD proteins alone and in complexes with Cul3. Right – small angle X-ray scattering of indicated KCTD proteins in complex with Cul3. Data are shown in green and scattering calculated from the inset model shown as a solid black line (BTBs - blue cartoon; Cul3 – yellow cartoon). (A) Data collected using the multi-domain KCTD17^BTB+CTD construct. Chi value for SAXS fit – 2.9 (B) Data collected using the BTB domain of SHKBP1. Chi value for SAXS fit – 2.2. (C) Data collected using the BTB domain of KCTD13. KCTD13 was prone to aggregation (as indicated by the purple line in the size-exclusion chromatography). A further size-exclusion chromatography step was needed for homogeneity (light blue line). Chi value for SAXS fit – 2.1. (D) Size-exclusion chromatograms (UV 280 nm) of KCTD16 alone and in the presence of Cul3 showing no apparent binding.

Figure 7.. Model of Cul3 binding to KCTD proteins.

(A) View of the known KLHL11-Cul3 structure highlighting the interface positions of the KLHL11 BTB (red) and 3-box (light blue) domains (PDB ID 4AP2) [10]. Other C-terminal KLHL11 regions are shown in grey while Cul3 is shown in green. (B) Homology model of the KCTD17–Cul3 interface based on the conserved BTB domain regions of KCTD17 and KLHL11. The Cul3 subunit (coloured green) binds to the equivalent BTB domain of KCTD17 (red), but also forms additional contacts with an adjacent BTB domain (yellow) in the KCTD17 pentameric ring. (C) Model of the KCTD17 ubiquitin ligase complex with charged E2-ubiquitin pairs. Remaining parts of the complex were modelled from other homologous structures (PDB IDs 3DQV [13], 1LDK [11], 4AP4 [47]). An alternative view from the top of the complex is presented in Supplementary Figure S3.