Structure‐Guided Design of Peptides as Tools to Probe the Protein–Protein Interaction between Cullin‐2 and Elongin BC Substrate Adaptor in Cullin RING E3 Ubiquitin Ligases

Abstract

Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi‐subunit complexes that control the fate of many proteins in cells. CRLs are attractive drug targets for the development of small‐molecule inhibitors and chemical inducers of protein degradation. Herein we describe a structure‐guided biophysical approach to probe the protein–protein interaction (PPI) between the Cullin‐2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel‐Lindau complex (CRL2VHL) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the “EloC site”, with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2‐adaptor PPI within CRLs.

Surface Probing by Fragment-Based Screening and Computational Methods Identifies Ligandable Pockets on the von Hippel–Lindau (VHL) E3 Ubiquitin Ligase

“…Consistent with these predictions, an M105A EloC mutant resulted in a 35-fold decrease in binding affinity toward Cul2 compared to wild-type EloC, 48 and a K4A Cul2 mutant peptide, which would prevent a salt bridge between K4 Cul2 and D179 VHL , exhibited no binding to VCB. 42 The HIF-recognition site in VHL was properly detected by the mixed-solvent MD method. However, FTMap failed to locate it and SiteMap perceived it as undruggable, consistent with the observation that only one fragment targeting the HIF site emerged from our biophysical screen (SI, Figure S1 ) and with the challenges in detecting binding of fragments resulting from deconstructing ligands targeting this interface.…”

The Role of Cullin-RING Ligases in Striated Muscle Development, Function, and Disease

“…Interestingly, the association of Co 2+ ions does not abrogate the binding of elongin-B/C or the VHL substrate adapter, suggesting an allosteric inhibition of the E3-ligase activity [ 57 ]. A more recent approach to modulating cullin-2 activity is based on short peptide sequences that disrupt the binding of cullin-2 to the elongin-B/C heterodimer ( Table 1 ) [ 58 ]. However, this approach is currently marred by challenges, such as low affinity or the complex structural nature of the protein interaction network required to form cullin-2-based E3-ligases.…”

Targeting Uracil-DNA Glycosylases for Therapeutic Outcomes Using Insights from Virus Evolution

“…Looking at the HIV-1 accessory protein Vpr, its interactions with UNG2 show a different bonding pattern in the Ung DNA binding cleft, supported by extra interactions on the surface around it [21]. Again, this could provide more clues as to how to build a novel chemical entity which effects uracil-free inhibition of Ung, as in effect this structural insight provides some pre-prepared peptide mapping [113]. There are PPI interfaces to define, other than Vpr, which could be sites for important new UNG2 targeting drug approaches against HIV.…”

“…Considering also, PPIs: disrupting them with novel chemical entities may progress from mapping out druggable patches at the contact interfaces using peptides [113]. This strategy in itself may result in a drug-like molecule, since peptidic drugs are successful in their own right (as reviewed in [114]).…”