Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases^*

Protein Expression and Purification

Human KLHL11 (UniProt Q9NVR0, residues 67–340), KLHL2 (O95198, residues 294–593), KLHL7 (Q8IXQ5, residues 283–586), and KLHL12 (Q53G59, residues 268–567) were subcloned into the vector pNIC28-Bsa4. Human Cul1_NTD (Q13616, residues 1–412), Cul3_NTD (Q13618, residues 1–390), Cul3_NTDΔN22 (residues 23–390), Cul5_NTD (Q93034, residues 1–386), and KBTBD5 (Q2TBA0, residues 314–621) were subcloned into the vector pNIC-CTHF using ligation-independent cloning, as described previously (31). Two point mutations were introduced into the Cul1 (V367R and L371D), Cul3 (I342R and L346D), and Cul5 (V341R and L345D) sequences to stabilize the isolated N-terminal domains. Proteins were expressed in BL21(DE3)-R3-pRARE cells using 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for overnight induction at 18 °C. Harvested cells were resuspended in binding buffer (50 mm HEPES, pH 7.5, 500 mm NaCl, 5% glycerol, 5 mm imidazole) supplemented with 1 mm phenylmethylsulfonyl fluoride and 1 mm tris(2-carboxyethyl)phosphine and disrupted by sonication or high pressure homogenization. Proteins were purified by nickel affinity and size exclusion chromatography (proteins for complexes were mixed 1:1 after IMAC). A final ion exchange step was used for KLHL7 (Mono S) and the KLHL11-Cul3 complexes (HiTrap Q). Tobacco etch virus protease A was used to cleave the polyhistidine tags from KLHL2, KLHL7, KLHL12, and KBTBD5 overnight at 4 °C. Selenomethionine-substituted KLHL11 was prepared using M9 minimal growth media supplemented with selenomethionine during the exponential growth phase.

Crystallization and Data Collection

Crystals were grown using the sitting-drop vapor-diffusion technique and cryoprotected before being vitrified in liquid nitrogen (full conditions are listed in Table 1). Diffraction experiments were conducted at 100 K. Data were collected using synchrotron radiation with the exception of KLHL7, for which a full dataset was collected using a Rigaku FR-E SuperBright rotating-anode x-ray generator. Crystallographic data are provided in Table 2. To calculate experimental phases, datasets were collected from crystals of selenomethionine-substituted KLHL11 at a wavelength of 0.9794 Å and from crystals of the KLHL11-Cul3_NTDΔN22 complex soaked with 2 mm thimerosal at a wavelength of 0.9686 Å.

Structure Determination

Data were integrated using Mosflm (32) or XDS (33) and scaled with SCALA (34) or AIMLESS as part of the CCP4 software suite (35). Experimental phases were calculated, and density modification was carried out with autoSHARP (36). Alternatively, phases were calculated using molecular replacement with PHASER (37) and density modification conducted with PARROT (38). Automated model building tasks were conducted with BUCCANEER (39, 40) or PHENIX.AUTOBUILD (41). Manual model building was performed with COOT (42) and the models refined with CNS (43, 44), REFMAC (45, 46), or BUSTER (47) using TLS and NCS restraints as appropriate. Experimental phase restraints were included in the refinement of the KLHL11-Cul3_NTDΔN22 complex until the final round of refinement. The model of this Cul3 complex was used as a molecular replacement solution for the higher resolution KLHL11-Cul3_NTD structure. The final model was completed manually and refined to completion. Models were validated using the PHENIX (41) validation tools and/or MOLPROBITY (48).

Isothermal Titration Calorimetry

ITC experiments were performed at 15 °C using a Microcal VP-ITC microcalorimeter. Proteins were dialyzed into a buffer containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 0.5 mm tris(2-carboxyethyl)phosphine. Cullin proteins (90–125 μm) were titrated into KLHL11 (10 μm). Data were analyzed using a single binding site model implemented in the Origin software package provided with the instrument.

BTB-Kelch Family

The human genome contains some 52 BTB-Kelch family proteins (Fig. 1 and supplemental Table S1). Their nomenclature is varied, but the protein family can be subdivided into the Kelch-like (KLHL(1–39)) and the Kelch repeat and BTB domain-containing proteins (KBTBD(1–13)). To derive structural models for their function as Cul3-based E3 ligases, members of this family were subcloned and screened for bacterial expression. Soluble expression constructs were identified that contained the BTB-BACK or Kelch domains but not the BACK-Kelch or BTB-BACK-Kelch domains. Crystal structures of the BTB-BACK domains of human KLHL11 were subsequently solved alone and in complex with the N-terminal Cullin-repeat domain of Cul3. Additional structures were solved of the Kelch domains of human KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5 (see Table 1 for crystallization conditions). A summary of statistics for data collection and refinement is reported in Table 2.

Structure of the BTB-BACK Domains of KLHL11

The structure of the BTB-BACK domains of KLHL11 (Fig. 2A, residues 67–340) was determined at 2.6-Å resolution using phases calculated from single-wavelength anomalous diffraction collected from selenomethionine-incorporated protein crystals. Two protein chains were present in the asymmetric unit forming a homodimer with an elongated shape of overall dimensions 150 × 35 × 25 Å (Fig. 2B). The BTB domain of KLHL11 closely resembles the common BTB fold of promyelocytic leukemia zinc finger protein (PDB code 1BUO, root mean square deviation 4.65 Å for 118 Cα atoms) (9). An interchain β1–β5′ antiparallel β-sheet establishes the typical domain-swapped dimer, which is additionally stabilized by the interactions between helices α1 and α2′ (where ′ denotes the second chain) (Fig. 2C).

Portions of the all α-helical BACK domain, also known as the intervening region, have been previously structurally determined for Gigaxonin (KLHL16) (30) and KBTBD4 (PDB code 2EQX). KLHL11 is the first structure to span the entire domain and contains eight helices in total. The two N-terminal helices (KLHL11 α7–α8) make up the 3-box motif and bind helices α5 and α6 of the BTB domain in an antiparallel four helix bundle configuration (Fig. 2C). Significantly for Cul3 recognition, all three structures are closely conserved despite limited sequence identity. The remaining C-terminal helices α9–α14 form a distinct subdomain, packing perpendicular to the 3-box. Notably, this arrangement creates a significant cleft some 16 Å deep and 18 Å wide between the BTB and BACK domains that exposes the 3-box for Cullin interaction (Fig. 2B).

Because the BTB-Kelch proteins are single chain analogs of other CRL substrate adaptors, such as Skp1/β-TrCP1, the BACK domain was expected to fold similarly to the F-box and helical linker regions of β-TrCP1 (15, 49). However, superposition of KLHL11 reveals a significant deviation from this arrangement (Fig. 2D). In particular, the long C-terminal helices that support the substrate-binding WD40 (β-TrCP1) or Kelch (KLHL11) domain show a 50° change in orientation (Fig. 2D). In part, this results from the distinct orientation of the 3-box, which packs perpendicular to the equivalent F-box or SOCS box motifs of other CRL substrate-recognition domains (Fig. 2E). These changes likely reflect the distinct architecture of the CRL3 class imposed by the BTB dimer.

Structural Diversity of the Kelch Substrate-Recognition Domain

The Kelch domain is the most widespread of the CRL3 substrate-recognition domains and recruits a diverse range of substrates. Structures are previously known only for the Kelch-like protein KEAP1 (KLHL19) (Fig. 3A) (22, 50, 51) and the Kelch-related protein KBTBD10 (KRP1) from Rattus norvegicus (52). To further characterize the diversity of these substrate-recognition domains, we determined the Kelch domain structures of the Kelch-like proteins KLHL2, KLHL7, and KLHL12, as well as the Kelch-related protein KBTBD5. All structures were refined at high resolution, ranging between 1.6 and 2.0 Å.

Although all four proteins display the typical β-propeller structure (Fig. 3B), there are systematic differences between KLHL and KBTBD proteins. The six Kelch repeats form the six “blades” of the propeller, each consisting of a four-stranded antiparallel β-sheet (Fig. 3A), with a C-terminal βA strand closing the propeller by completing blade I. This site shows divergence between the KLHL and KBTBD proteins. The Kelch-like (KLHL) proteins are characterized by consensus repeats, including a Gly-Gly pair that terminates strand βB, and hydrophobic Tyr (βC) and Trp (βD) residues that pack between blades (supplemental Fig. S1). In contrast, KBTBD5 exhibits an atypical repeat resulting in a more twisted blade I structure, as observed previously for KBTBD10 (52). Both KBTBD proteins also possess an extended C terminus that contributes a short βE strand to blade I as well as the usual βA (Fig. 3). Sequence comparisons suggest that this altered structure is a common feature of the KBTBD proteins (supplemental Fig. S1).

The surface properties of the six structures are strikingly diverse, reflecting their limited sequence identity. The substrate-binding site, first identified in the KEAP1-Nrf2 complex (51), lies on the narrower upper face of the Kelch domain (Fig. 3A). This surface displays a distinct electrostatic potential in each structure (Fig. 3B). The shape and size of the substrate pocket is determined by the inter-blade DA loops as well as the variable BC loops, which protrude significantly out of the upper propeller face (Fig. 3, A and C). Interestingly, KBTBD5 and KBTBD10 display a rather atypical loop arrangement, with an unusually short BC loop (three residues) in blade I flanked by greatly extended loops (18 and 20 residues, respectively) in blades II and VI (Fig. 3C). By contrast, the BC loops in the KLHL structures are more evenly distributed, with an average loop length of 12 residues. These variant features are expected to contribute to the precise substrate specificity of each BTB-Kelch family member.

Cul3 Binding to the 3-Box Involves a Specific N-terminal Extension Sequence

Substrate ubiquitylation requires the BTB-Kelch proteins to assemble with Cul3. To establish the molecular basis for Cul3 interaction, we adopted a split and express strategy for soluble protein expression (53). The Cul3 NTD was subcloned with the solubilizing mutations I342R/L346D and co-purified with the previously characterized BTB-BACK domains of KLHL11. Crystals of the complex were first obtained in space group C2 using a Cul3 construct containing only the three consecutive Cullin-repeat domains (Cul3_NTDΔ22, Fig. 4A). Diffraction data were collected to 3.1-Å resolution, and the structure was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. The structural model indicated the potential for an unexpected interaction from the deleted Cul3 N-terminal extension. To address this, we identified a new crystal form in space group I121 containing the full Cul3_NTD (Fig. 4A). The structure of this complex was solved using molecular replacement and refined at 2.8-Å resolution (Fig. 4B). Importantly, the electron density map allowed unambiguous tracing of a further eight N-terminal residues from Cul3 that packed in the hydrophobic groove located with the 3-box between the BTB and BACK domains of KLHL11 (Fig. 4C). An analogous groove was identified in the SCF complexes of Skp1 but was bound to an N-terminal sequence preceding the F-box (10, 49).

We were interested to assess the contribution of these Cul3 residues to the interaction and therefore measured the binding affinity of different Cullin proteins by ITC. Cul3_NTDΔ22 containing only the Cullin-repeat domains bound KLHL11 relatively weakly (K_D = 0.65 μm, Fig. 4D). However, binding affinity was increased 30-fold upon inclusion of the Cul3 N-terminal extension (Cul3_NTD; K_D = 20 nm, Fig. 4D). As expected, there was essentially no binding of KLHL11 to the Cul1_NTD or Cul5_NTD (Fig. 4D), demonstrating the specificity of the interaction.

Structure of the KLHL11-Cul3 Complex

The two crystallized KLHL11-Cul3 complexes exhibited a 2-fold symmetry axis across the BTB dimer, resulting in only one KLHL11 and one Cul3 chain in each asymmetric unit. Generation of the symmetry-related molecules revealed the expected heterotetrameric assemblies, with each subunit in the KLHL11 homodimer binding one molecule of Cul3 (Fig. 4B). KLHL11 was clearly defined by electron density between residues 67 and 336 and the Cul3_NTD between residues 17 and 381. Overall, the contact surface area of the Cul3_NTD interface (1508 Ų) was significantly larger than that of the Cul3_NTDΔ22 complex (1018 Ų), highlighting the importance of the N-terminal extension for Cul3 binding (Figs. 5, A and B, and 6A). By comparison, the SPOP-Cul3 structure (12), which lacks both the N-terminal extension and the 3-box/BACK domain, had a contact area of just 830 Ų. Despite its stalk-like appearance, the Cul3 structure is remarkably conserved between the SPOP and KLHL11 complexes (0.8–1.3 Å root mean square deviation over 300 Cα atoms), suggesting that its conformation is relatively rigid.

The primary interface in the Cul3 complexes is formed by helices H2 and H5 of the first Cullin repeat, which pack against the BTB and 3-box domains of KLHL11 (Fig. 6B). Sequence conservation in the H2 and H5 helices is strong between Cul3 orthologs but weaker between paralogs (7). Furthermore, the H2 helix of Cul3 is a turn shorter than either Cul1 (7) or Cul5 (PDB code 2WZK) and therefore inserts comfortably into a shallow cleft in the BTB surface (Fig. 4C). In both KLHL11 and SPOP, this cleft forms via an induced fit mechanism facilitated by conformational changes in the α3-β4 loop (Fig. 7). This loop is disordered, or associated with higher crystallographic B-factors, in the unbound crystal structures but becomes helical upon Cul3 binding enabling hydrogen bonds from KLHL11 Ser-131 and Glu-132 to the Cul3 H2 helix (Figs. 6B and 7). In addition, Phe-130 (SPOP Met233) is shifted by some 5 Å to insert into a deep hydrophobic pocket formed between Cul3 helices H2 and H4 (Figs. 6B and 7).

The KLHL11 complex reveals for the first time how the 3-box binds to Cul3. The motif, including KLHL11 α7 and α8, contributes both to the interface with the first Cullin repeat (Fig. 6B) and to the hydrophobic groove that accommodates the Cul3 N-terminal extension sequence (Fig. 6A). The Cullin-repeat interaction is mediated largely by the 3-box α7-α8 loop that packs against the C-terminal regions of Cul3 H2 and H5 (Fig. 6B). Here, the backbone carbonyl of KLHL11 His-213 (α7) forms a single hydrogen bond with Cul3 Lys-68. The Cul3 N-terminal extension sequence runs antiparallel to the 3-box α7 helix and is flanked by the α5/α6 and α9/α10 helices of the BTB and BACK domains, respectively (Fig. 6A). A single hydrogen bond is formed from the side chain oxygen of Cul3 Thr-24 to the backbone nitrogen of KLHL11 Phe-246 (α10). The remaining N-terminal Cul3 contacts are hydrophobic. In particular, the side chains of Cul3 Ile-18, Ala-20, and Pro-22 insert directly into the hydrophobic groove of KLHL11 to contribute substantially to the overall contact area (Fig. 5A).

Model for the Dimeric CRL3 Complex

The KLHL11-Cul3 structure establishes the core architecture of the dimeric BTB-Kelch class of E3 ligase. To generate a working model of the complete E3, we built the missing structural domains using other available structures. The Cul3_CTD was built initially from the Cul1 structure (PDB 1LDK) (7) and then modified to fit the active conformation of the neddylated Cul5_CTD-Rbx1 complex (PDB 3DQV) (5). An E2-ubiquitin intermediate was modeled from the UbcH5A-ubiquitin structure (PDB 4AP4) (54) and docked onto Rbx1 by its homology to the Cbl-UbcH7 complex (PDB 1FBV) (55). Finally, the Kelch domain and substrate from the KEAP1-Nrf2 complex (PDB 2FLU) (51) were modeled atop helix α14 of the BACK domain of KLHL11. The final model places the two E2-ubiquitin intermediates at the center of the complex where they dissect the axis between the two Kelch domains (Fig. 8A). Flexibility in the linker between the BACK and Kelch domains as well as the limited freedom of the RING domain of Rbx1 may help to bridge the substrate-ubiquitin gap and to break the overall symmetry specified by KLHL11.