Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases

Abstract

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

FIGURE 1.

Phylogenetic tree of human Kelch domains from the BTB-Kelch family. A, multiple sequence alignment of human Kelch domains was generated using ClustalX (version 1.83) (65) and manually refined with reference to publicly available structures. A phylogenetic tree was created from this alignment using the N-J Tree export functionality of ClustalX and a radial tree figure prepared in PhyloDraw (version 0.8) (66). Further descriptions of each protein are given in supplemental Table S1.

FIGURE 2.

Structure of the BTB-BACK domains of human KLHL11. A, domain organization of human KLHL11. The domain boundaries of the BTB, BACK, and Kelch domains of KLHL11 are indicated, as well as the six Kelch repeats that constitute the complete Kelch domain. B, surface representation of the dimeric KLHL11 structure. Functional domains are color-coded in chain A, and chain B is colored gray. The 3-box includes the first two helices of the BACK domain. C, ribbon diagram of the of KLHL11 structure colored as in B and labeled by the convention of promyelocytic leukemia zinc finger protein (9). D, similar fold of the BTB domain and Skp1 was used for superposition of KLHL11 and the Skp1/β-TrCP1 complex (PDB code 1P22) (49). The BACK domain shows a different fold to the helical linker of β-TrCP1. Dashed lines mark the different orientations of the C-terminal helices that support the respective Kelch and WD40 β-propeller domains. E, superposition reveals that the 3-box of KLHL11 folds perpendicular to the analogous F-box of β-TrCP1 as well as the SOCS box of SOCS4 (PDB code 2IZV) (67).

FIGURE 3.

Structural diversity of the Kelch domains. A, superposition of the Kelch domains of KLHL2, KBTBD5, and the KEAP1-Nrf2 complex (PDB code 2FLU) (51). The six Kelch repeats, forming the “blades” of the β-propeller, are numbered I–VI from the N terminus. A schematic of one repeat shows the four constituent β-strands labeled A–D and the variable BC loop that contributes to substrate recognition. Three highly distinct BC loop conformations are marked *1–*3 in the ribbon diagram and similarly labeled in C. B, ribbon and surface representations of the Kelch domains of human KLHL2, KLHL7, KLHL12, and KBTBD5 as well as the previously solved structures of KEAP1 (PDB code 2FLU) and KBTBD10 (KRP1) from R. norvegicus (PDB code 2WOZ) (52). Electrostatic surface potentials are colored on a scale between −10 kT/e (red) and +10 kT/e (blue). A dashed line surrounds the unusual βE strand in the KBTBD proteins. C, schematic comparison of BC loop lengths across the six blades of each structure (lengths are indicated in white). Three loops marked in A are similarly labeled *1–*3 for reference.

FIGURE 4.

Structure of the KLHL11-Cul3 complex. A, domain organization of human Cul3 showing the domain boundaries of the N-terminal Cullin repeats and the C-terminal domain. The sequence coverage of the Cul3_NTD and Cul3_NTDΔ22 constructs used for crystallization and ITC is indicated below. B, ribbon representation of the human KLHL11-Cul3 structure highlighting different functional domains. C, surface representation of KLHL11 showing the interface with Cul3 (cylinder representation) as viewed from below with respect to B. Residues preceding Cul3 H1 (the N-terminal extension) bind the 3-box in a hydrophobic groove located between the BTB and BACK domains and are shown as sticks with the corresponding electron density (2F_o − F_c map, contoured at 1σ) shown in blue. The structures of Cul1 (PDB code 1LDK) (7) and mouse Cul5 (PDB code 2WZK) are superimposed revealing the shorter H2 helix in Cul3. D, ITC measurements of the binding of KLHL11 to different human Cullin N-terminal domains demonstrating the absence of binding to non-Cul3 domains.

FIGURE 5.

N-terminal extension contributes significantly to the contact surface area. A, residue-based surface areas of Cul3_NTD and Cul3_NTDΔ22 buried by KLHL11 interaction are shown as bar graphs. Values were calculated using the protein interfaces, surfaces, and assemblies service at the European Bioinformatics Institute (68). B, residue-based surface areas of KLHL11 buried by Cul3_NTD and Cul3_NTDΔ22, respectively, are shown as bar graphs.

FIGURE 6.

Specific interactions in the KLHL11-Cul3 interface. A, side chain interactions of the Cul3 N-terminal extension sequence in the hydrophobic groove of KLHL11. Intermolecular hydrogen bonds are shown by a dashed blue line. The side chains of Arg-17 and Arg-19 were not clearly defined in the electron density and were not built. View shown is the same as Fig. 4C. B, side chain interactions of the Cullin repeat domain with the BTB and 3-box domains of KLHL11 (same view as above).

FIGURE 7.

Conserved assembly of SPOP-Cul3 and KLHL11-Cul3 complexes. A, superposition of the unbound (magenta) and bound (gray) KLHL11 structures highlighting the conformational change of the α3–β4 loop upon association with Cul3 (orange). A dashed arrow indicates a 5-Å movement of KLHL11 Phe-130. B, superposition of the unbound and bound SPOP structures highlights a similar conformational change of the α3-β4 loop upon association with Cul3. A dashed arrow indicates the movement of Met-233 (equivalent to KLHL11 Phe-130).

FIGURE 8.

Model of an active BTB-Kelch E3 ligase. A, model of a complete BTB-Kelch E3 ligase complex was constructed using the core architecture defined by the KLHL11-Cul3 complex. Missing structural domains were modeled from other available structures, including PDB codes 1LDK and 3DQV for the Cullin CTD-Rbx1-Nedd8 complex, 1FBV and 4AP4 for the E2-ubiquitin intermediate, and 2FLU for the Kelch-substrate complex. Asterisks mark the positions of the reactive E2-ubiquitin (Ub) thioester bonds. B, schematic representation of the two-site recognition model proposed for Nrf2 recruitment by KEAP1. The intervening α-helix contains seven substrate lysines of which six are predicted to fall on the same face (18).