Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

Abstract

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21

“…Indeed, we have recently reported structures of Ubvs that target E3 ligases of the HECT (24), SCF (25), and RING families (43). Thus, the methods described here can be applied to dissect the functional epitopes for Ubvs targeting many other components of the Ub proteasome system.…”

“…Moreover, interpretation of the results in the context of the human USP family shows that many conserved functional interactions likely are generalizable to most family members. We have recently reported structures of additional Ubvs in complex with E3 ligases of the Homologous to E6AP C terminus (HECT), Skp1 Cul1 F-box (SCF), and Really Interesting New Gene (RING) families (24,25), and the extension of the methods described here to these and other types of Ub-protein interactions may identify commonalities and differences in how Ub recognizes diverse structural folds to mediate biological effects.…”

Flexibility and Design: Conformational Heterogeneity along the Evolutionary Trajectory of a Redesigned Ubiquitin

“…Directed evolution can stabilize the extra states introduced by the mutations selected through protein design, quenching heterogeneity. Given the recent interest in using ubiquitin variants for structural biology chaperones as in vitro modulators of the ubiquitin proteasome system (Canny et al, 2016; Ernst et al, 2013; Gorelik et al, 2016; Phillips et al, 2013; Zhang et al, 2016), there may be additional opportunities to use these proteins to test these hypotheses and learn about the importance of conformational dynamics in protein function (Phillips et al, 2013). Experimentally characterizing nearly iso-energetic states will improve our ability to evaluate the success of the design of protein ensembles.…”

“…Protocol adapted from Zhang et al ((Canny et al, 2016; Ernst et al, 2013; Gorelik et al, 2016; Phillips et al, 2013; Zhang et al, 2016)) and references therein. Each of the ubiquitin mutants were expressed from a pET derivative vector containing the protein gene with a TEV cleavable N-terminal His6 tag in E. coli BL21(DE3) cells.…”

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha‐helical ubiquitin interacting motif

“…Design and implementation of phage‐displayed libraries to identify UbVs that bind with enhanced affinity to specific targets has been a boon to such research. Recent applications of this approach have been successful in producing UbVs capable of modulating particular biological functions either as inhibitors or activators of Ub enzymes . Here, we have extended this body of work by applying the UbV technology to the UIM subfamily of UBDs.…”