Decoding the selective chemical modulation of CYP3A4
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Miller, Darcie
Publications and source records attributed to Miller, Darcie.
Structural basis for C-degron selectivity across KLHDCX family E3 ubiquitin ligases
Abstract Specificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the termini of substrates. However, our knowledge of structural features distinguishing closely related C-degron substrate-E3 pairings is limited. Here, by systematically comparing ubiquitylation activities towards a suite of common model substrates, and defining interactions by biochemistry, crystallography, and cryo-EM, we reveal principles of C-degron recognition across the KLHDCX family of Cullin-RING ligases (CRLs). First, a motif common across these E3 ligases anchors a substrate’s C-terminus. However, distinct locations of this C-terminus anchor motif in different blades of the KLHDC2, KLHDC3, and KLHDC10 β-propellers establishes distinct relative positioning and molecular environments for substrate C-termini. Second, our structural data show KLHDC3 has a pre-formed pocket establishing preference for an Arg or Gln preceding a C-terminal Gly, whereas conformational malleability contributes to KLHDC10’s recognition of varying features adjacent to substrate C-termini. Finally, additional non-consensus interactions, mediated by C-degron binding grooves and/or by distal propeller surfaces and substrate globular domains, can substantially impact substrate binding and ubiquitylatability. Overall, the data reveal combinatorial mechanisms determining specificity and plasticity of substrate recognition by KLDCX-family C-degron E3 ligases.
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Principles of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2
Abstract PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2’s U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3 BD2 . Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.
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