Recombinant VCB Trimeric complex: VHL(M54-D213):EloC(M17-C112):EloB(M17-C112)

The three proteins that form the VCB complex were expressed in parallel under three different promoters on two plasmids. The VCB complex is formed by von Hippel–Lindau protein (VHL) spanning residues G_M54-D213 with an additional N-terminal G remaining from the TEV cleavage. VHL forms a complex with ElonginB (M1-K104) and ElonginC (M17-C112).

Protein Construct: G_VHL(M54-D213):EloC(M17-C112):EloB(M17-C112)
Source: Human
Expression Host: Escherichia coli
Molecular weight [kDa]: 41.1
pI: 5.92
Extinction Coefficient [M^-1cm^-1]: 25900
Concentration: 10.2 mg/mL
Storage Buffer: 20 mM HEPES; pH 7.5, 100 mM NaCl, 1 mM DTT
Tag: none

Amino acid sequence:

QC Data: Datasheet
Purity SDS: >95 %
Hydrodynamic Radius (nm): 3.2
Polydispersity Index (PDI): 0.11
T_on (°C): 26.8
T_m (°C): 43.9

Structure and function

VCB is the substrate receptor of the Cullin-RING E3 ubiquitin ligase CRL2^VHL and plays an important role in the Oxygen-Sensing pathway by regulating transcription factor hypoxia-inducible factor (HIF)-1ɑ through ubiquitin transfer and proteasomal degradation. The full E3 ligase is a large, pentameric protein complex build up by trimeric VCB, the scaffold protein Cullin-2 and the RING finger subunit Rbx1.
The VCB complex itself consists of the von Hippel-Lindau protein (VHL) and the two adaptor proteins ElonginC (EloC) and ElonginB (EloB). VHL serves as the central substrate binding module and features a hydroxyproline (Hyp) substrate recognition site as well as a binding domain that interacts with EloC. The binding of VHL to the N-terminal region of Cullin-2 and the stabilization of the complex is mediated by the two adaptor proteins EloC and EloB.

Figure 1: Architecture of the Cullin-RING E3 ligase system CRL2VHL with VCB binding to Cullin-2. Adapted from (Cardote et al., 2017)

VCB Success in Smart-Soak Campaign

VCB serves as a critical component in PROTAC design by recruiting the target proteins via its central substrate binding module VHL. Formation of a ternary complex involving the target protein is required for targeted protein degradation (TPD). There is a need for novel chemical matter for recruitment of target proteins via VHL. Enhanced molecular characteristics of next-generation VHL binders could broaden the efficacy of VCB-mediated TPD.

After crystallizing the VCB complex, a SmartSoak system was set up and it was observed that SmartSoak improves the diffraction of the crystal by 0.5 Ångström. Subsequently, an in-house fragment library was SmartSoak'ed and novel VCB binders could be identified: Four binders at EloC, one binder at the EloC-EloB interface (cryptic pocket) and three binders at VHL (cryptic and orthosteric).

Interestingly, two fragments bind at the cryptic pocket of VHL, inducing a more pronounced opening of the pocket compared to the published structures. The two fragments are well-positioned for linking.

Figure 2: SmartSoak® -enabled soaking system identifies allosteric and orthosteric binders of the VCB complex.