Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex

Abstract

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2^VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting.

Keywords: Cullin-2; RING domain proteins; VHL; cullin-RING E3 ubiquitin ligases; protein-protein interactions.

Graphical abstract

Figure 1

Crystal Structure of the CRL2^VHL Complex (A) Diagrams of the primary structure of each protein subunit, illustrating the constructs used to obtain the crystal structure. (B) Representation of the 2F_o-F_c electron density contoured at 1σ over a portion of the model. (C) Crystal structure of the CRL2^VHL with each of the protein subunits identified. Cul2 is divided into N-terminal (NTD) and C-terminal (CTD) domains and the NTD is composed of three helical bundles, constituted by five α-helices (A–E). (D) The three helical bundles of the NTD are superposable with a maximum RMSD of 4.5 Å over the Cα. (E) CTD of Cul2 is organized in a four-helical bundle (4HB), an α/β domain, and a winged-helix motif (WH-B).

Figure 2

The CRL2^VHL Complex Is Dynamic and Flexible (A) Crystal structures of the full NTD of Cul1 (PDB: 1U6G), Cul2 (PDB: 5N4W), Cul3 (PDB: 4HXI), Cul4A (PDB: 4A0K), Cul4B (PDB: 4A64), and Cul5 (PDB: 4JGH), illustrating the existence of hinge points in the linkers between Cullin repeats. (B) Full-length structures of Cul2, Cul4A, and Cul1 superposed by the CTDs reveal considerable inter-domain flexibility through a hinge point between NTD and CTD. (C) Close-up view of the CTDs of Cul1, Cul2, and Cul4A with the proteins aligned by the third Cullin repeat of the NTD, illustrating the different relative orientations of the CTDs in the full-length structures.

Figure 3

Rbx1 Presents a New Orientation Superposition of the CTDs of six Cullins—Cul1 (PDB: 1U6G), Cul2 (PDB: 5N4W), Cul4 (PDB: 4A0C), Cul5 (PDB: 3DPL), Cul5∼NEDD8 (PDB: 3DQV), and Glomulin-Rbx1-Cul1 (PDB: 4F52)—complexed with Rbx1. The new structure of Rbx1 in complex with Cul2 unveils a novel orientation of its RING domain, resembling an en route conformation between Rbx1-Cul5 and Rbx1-Cul5∼NEDD8.

Figure 4

The Cul2-VBC Interface (A) Contacts between Cul2_NTD and VBC. As in similar CRL complexes, helix α2 of Cullin is the closest to the adaptor and substrate receptor subunits, establishing hydrophobic contacts. (B) The N-terminal tail of Cul2 plays a substantial role in the PPI. Leu3 is accommodated in a hydrophobic pocket on the surface of EloC, and Pro5 is involved in a three-way interaction between Val181 from pVHL and Met105 from EloC. (C) Residues Asp187 and Lys159 in pVHL establish an electrostatic network with residues Lys114 and Gln111 in helix α5 of Cul2. (D) Residues Asn36, Phe39, Tyr43, and Val47 participate in hydrophobic contacts at the interface between helix α2 and EloC and pVHL.

Figure 5

Biophysical Characterization of the Interaction between VBC and Cul2 (A) ITC data whereby VBC (200 μM) was titrated into Rbx1-Cul2 (20 μM) at 303 K. Under these conditions, the binding affinity of the interaction (K_D) is 42 nM. (B) Temperature dependency of the thermodynamic parameters ΔH, ΔG and −TΔS. The change in heating capacity, ΔC_p, was derived from the change in enthalpy with the temperature. (C) Comparison of ΔC_p values for similar CRL systems.

Figure 6

Residues in the EloC Hydrophobic Pocket Reveal Criticality for the Strong Binding Affinity of Cul2-VBC (A) Plot of the log(IC₅₀) values resulting from the AlphaLISA competition experiment where mutant constructs of VBC or Cul2 were used to displace the native interaction between bead-bound wild-type protein. Mutations at the EloC pocket are highlighted in yellow, mutations at helix α5 interface are highlighted in purple, and mutations at helix α2 interface are highlighted in green. The fitting and calculation of IC₅₀ values was performed with GraphPad Prism 7 software, and the error bars represent the error in the fitting function. (B) Dose-response curves of the raw AlphaLISA data showing representative displacers from the three interaction areas in comparison with VBC wild-type (wt). The experiments were performed in quadruplicate and the results are an averaged value. The error bars represent the SD of each point.

Figure 7

Swapping Residues and Selectivity for Cul2 or Cul5 (A) Structures of Cul5-SBC and Cul2-VBC aligned by the EloC subunit show residues involved in the electrostatic network created between substrate receptor and Cullin, in both cases. (B) AlphaLISA data show loss of binding affinity of V^QRYBC toward Cul2 and rescue of binding of S^KSDBC toward Cul2. The experiments were performed in quadruplicate and the results are an averaged value. The error bars represent the SD of each point. The fitting was performed with GraphPad Prism 7 software. (C and D) ITC data. Titrant solution (200 μM) was diluted into 20 μM titrate over 19 injections of 2 μL at 303 K. (C) Titration of SBC and S^KSDBC into Rbx1-Cul2. (D) Titration of Cul5_NTD into V^QRYBC and VBC. (E) Summary of the results obtained in the biophysical experiments.